Technical Field
[ 1] The present disclosure provides T -cell modulatory multimeric polypeptides
[2]
(TMMPs) that comprise an immunomodulatory polypeptide and that comprise an
epitope-presenting Wilms tumor peptide.
Background Art
[3] An adaptive immune response involves the engagement of the T cell receptor (TCR),
[4]
present on the surface of aT cell, with a small peptide antigen non-covalently
presented on the surface of an antigen presenting cell (APC) by a major histocompatibility
complex (MHC; also referred to in humans as a human leukocyte antigen
(HLA) complex). This engagement represents the immune system's targeting
mechanism and is a requisite molecular interaction forT cell modulation (activation or
inhibition) and effector function. Following epitope-specific cell targeting, the targeted
T cells are activated through engagement of costimulatory proteins found on the APC
with counterpart costimulatory proteins the T cells. Both signals - epitopeffCR binding
and engagement of APC costimulatory proteins with T cell costimulatory proteins - are
required to drive T cell specificity and activation or inhibition. The TCR is specific for
a given epitope; however, the costimulatory protein not epitope specific and instead is
generally expressed on all T cells or on large T cell subsets.
Disclosure of Invention
Technical Problem
[5] The present inventors attempted to prepare T-cell modulatory multimeric
[6]
polypeptides useful for modulating the activity of aT-cell and for modulating an
immune response in an individual.
Solution to Problem
[7] In accordance with one aspect of the present disclosure, a T -cell modulatory
multimeric polypeptide is disclosed.
[8] In accordance with another aspect of the present disclosure, a nucleic acid
comprising the nucleic acid molecule is disclosed.
[9] In accordance with still another aspect of the present disclosure, an expression vector
comprising the nucleic acid is disclosed.
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[10] In accordance with still another aspect of the present disclosure, a method of selectively
modulating the activity ofT cell specific for a Wilms tumor-1 (WT-1) epitope
is disclosed.
[11] In accordance with still another aspect of the present disclosure, a method of
modulating an immune response in an individual is disclosed.
[12] In accordance with still another aspect of the present disclosure, a method of delivering
an immunomodulatory polypeptide selectively to a target T cell is disclosed.
[13] In accordance with still another aspect of the present disclosure, a method of
[14]
detecting, in a mixed population ofT cells obtained from an individual, the presence of
a target T cell that binds a WT -1 epitope is disclosed.
Advantageous Effects of Invention
[ 15] A T -cell modulatory multimeric polypeptide is useful for modulating the activity of a
T cell, and for modulating an immune response in an individual.
[16]
Brief Description of Drawings
[17] FIG. lA-lF are schematic depictions of various TMMPs of the present disclosure.
[18] FIG. 2A-2F are schematic depictions of various disulfide-linked TMMPs of the
present disclosure.
[19] FIG. 3A-3C provide amino acid sequences of exemplary polypeptide chains of
TMMPs of the present disclosure.
[20] FIG. 4A-4H provide amino acid sequences of immunoglobulin Fe polypeptides.
[21] FIG. 5 provides a multiple amino acid sequence alignment of beta-2 microglobulin
([32M) precursors (i.e., including the leader sequence) from Homo sapiens
(NP 004039.1; SEQ ID N0:267), Pan troglodytes (NP 001009066.1; SEQ ID
N0:267), Macaca mulatta (NP 001040602.1; SEQ ID N0:268), Bas taurus
(NP 776318.1; SEQ ID N0:269) and Mus musculus (NP 033865.2; SEQ ID N0:270).
Amino acids 1-20 are a signal peptide.
[22] FIG. 6 provides an amino acid sequence of full-length human A*2402 allele HLA
heavy chain.
[23] FIG. 7A-7B provide schematic depictions of double disulfide-linked TMMP of the
present disclosure.
[24] FIG. 8A-8C provide schematic depictions of examples of configurations of
disulfide-linked TMMPs of the present disclosure.
[25] FIG. 9 provide schematic depictions of examples of positions of immunomodulatory
polypeptides in TMMPs of the present disclosure.
[26] FIG. lOA-lOG provide amino acid sequences of exemplary polypeptide chains of
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TMMPs of the present disclosure.
[27] FIG. llA-llF provide amino acid sequences of exemplary polypeptide chains of
TMMPs of the present disclosure, in which the polypeptide chains comprise the WT-1
peptide RVPGV APTL (WT -1 302-310) (SEQ ID NO: SO).
[2S] FIG. 12A-12F provide amino acid sequences of exemplary polypeptide chains of
TMMPs of the present disclosure, in which the polypeptide chains comprise the WT-1
peptide RYPGVAPTL (WT-1 302-310; V303Y) (SEQ ID NO:S1).
[29] FIG. 13A-13F provide amino acid sequences of exemplary polypeptide chains of
TMMPs of the present disclosure, in which the polypeptide chains comprise the WT-1
peptide RYFPNAPYL (WT-1126-134) (SEQ ID NO:S2).
[30] FIG. 14A-14F provide amino acid sequences of exemplary polypeptide chains of
TMMPs of the present disclosure, in which the polypeptide chains comprise the WT-1
peptide RYPSCQKKF (WT-1 417-425; W41SY) (SEQ ID NO:S3).
[31] FIG. 15 depicts the effect of TMMPs, containing the WT1 peptide epitope 126-134
(M127Y) and HLA-A *24 heavy chains, on antigen-specific CDS+ T cell expansion.
[32] FIG. 16 depicts the effect of TMMPs, containing the WT1 peptide epitope WT1
302-310(V303Y) and HLA-A*24 heavy chains, on antigen-specific CDS+ T cell
expansiOn.
[33] FIG. 17A depicts the cytolytic activity ofWT1-specific T cells, expanded by
contacting cells with a TMMP containing the WT1 peptide epitope 126-134 (M127Y),
against target cells presenting native WT1 (126-134) peptides.
[34] FIG. 17B depicts the cytolytic activity ofWT1-specific T cells, expanded by
[35]
contacting cells with a TMMP containing the WT 1 peptide epitope WT 1
302-310(V303Y), against target cells presenting native WT1 (302-310) peptides.
Best Mode for Carrying out the Invention
[36] Definitions
[37] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to
a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
Thus, this term includes, but is not limited to, single-, double-, or multistranded
DNA or RNA, genomic DNA, eDNA, DNA-RNA hybrids, or a polymer
comprising purine and pyrimidine bases or other natural, chemically or biochemically
modified, non-natural, or derivatized nucleotide bases.
[3S] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein,
and refer to a polymeric form of amino acids of any length, which can include coded
and non-coded amino acids, chemically or biochemically modified or derivatized
amino acids, and polypeptides having modified peptide backbones.
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[39] A polynucleotide or polypeptide has a certain percent "sequence identity" to another
polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or
amino acids are the same, and in the same relative position, when comparing the two
sequences. Sequence identity can be determined in a number of different ways. To
determine sequence identity, sequences can be aligned using various convenient
methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.),
available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST,
ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/,
mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Bioi.
215:403-10.
[ 40] The term "conservative amino acid substitution" refers to the interchangeability in
proteins of amino acid residues having similar side chains. For example, a group of
amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine,
and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists
of serine and threonine; a group of amino acids having amide containing side chains
consisting of asparagine and glutamine; a group of amino acids having aromatic side
chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids
having basic side chains consists of lysine, arginine, and histidine; a group of amino
acids having acidic side chains consists of glutamate and aspartate; and a group of
amino acids having sulfur containing side chains consists of cysteine and methionine.
Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagineglutamine.
[41] The term "immunological synapse" or "immune synapse" as used herein generally
refers to the natural interface between two interacting immune cells of an adaptive
immune response including, e.g., the interface between an antigen-presenting cell
(APC) or target cell and an effector cell, e.g., a lymphocyte, an effector T cell, a
natural killer cell, and the like. An immunological synapse between an APC and a T
cell is generally initiated by the interaction of a T cell antigen receptor and major histocompatibility
complex molecules, e.g., as described in Bromley et al., Annu Rev
Immunol. 2001;19:375-96; the disclosure of which is incorporated herein by reference
in its entirety.
[ 42] "T cell" includes all types of immune cells expressing CD3, including T -helper cells
(CD4+ cells), cytotoxic T-cells (CDS+ cells), T-regulatory cells (Treg), and NK-T cells.
[43] The term "immunomodulatory polypeptide" (also referred to as a "co-stimulatory
polypeptide"), as used herein, includes a polypeptide on an antigen presenting cell
(APC) (e.g., a dendritic cell, a B cell, and the like) that specifically binds a cognate coimmunomodulatory
polypeptide on aT cell, thereby providing a signal which, in
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addition to the primary signal provided by, for instance, binding of a TCR/CD3
complex with a major histocompatibility complex (MHC) polypeptide loaded with
peptide, mediates a T cell response, including, but not limited to, proliferation, activation,
differentiation, and the like. An immunomodulatory polypeptide can include,
but is not limited to, CD7, B7-l (CD80), B7-2 (CD86), PD-Ll, PD-L2, 4-lBBL,
OX40L, Pas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular
adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB,
HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or
antibody that binds Toll ligand receptor and a ligand that specifically binds with
B7-H3.
[44] As noted above, an "immunomodulatory polypeptide" (also referred to herein as a
"MOD") specifically binds a cognate co-immunomodulatory polypeptide on aT cell.
[ 45] An "immunomodulatory domain" ("MOD") of a TMMP of the present disclosure
binds a cognate co-immunomodulatory polypeptide, which may be present on a target
T cell.
[ 46] "Heterologous," as used herein, means a nucleotide or polypeptide that is not found
in the native nucleic acid or protein, respectively.
[47] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA)
is the product of various combinations of cloning, restriction, polymerase chain
reaction (PCR) and/or ligation steps resulting in a construct having a structural coding
or non-coding sequence distinguishable from endogenous nucleic acids found in
natural systems. DNA sequences encoding polypeptides can be assembled from eDNA
fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic
acid which is capable of being expressed from a recombinant transcriptional unit
contained in a cell or in a cell-free transcription and translation system.
[48] The terms "recombinant expression vector," or "DNA construct" are used interchangeably
herein to refer to a DNA molecule comprising a vector and at least one
insert. Recombinant expression vectors are usually generated for the purpose of expressing
and/or propagating the insert(s), or for the construction of other recombinant
nucleotide sequences. The insert(s) may or may not be operably linked to a promoter
sequence and may or may not be operably linked to DNA regulatory sequences.
[ 49] As used herein, the term "affinity" refers to the equilibrium constant for the reversible
binding of two agents (e.g., an antibody and an antigen) and is expressed as a
dissociation constant (KD). Affinity can be at least 1-fold greater, at least 2-fold
greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least
6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at
least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold
greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least
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80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1,000-fold
greater, or more, than the affinity of an antibody for unrelated amino acid sequences.
Affinity of an antibody to a target protein can be, for example, from about 100
nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or
from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term
"avidity" refers to the resistance of a complex of two or more agents to dissociation
after dilution. The terms "immunoreactive" and "preferentially binds" are used interchangeably
herein with respect to antibodies and/or antigen-binding fragments.
[50] The term "binding," as used herein (e.g. with reference to binding of a TMMP to a
polypeptide (e.g., aT -cell receptor) on aT cell), refers to a non-covalent interaction
between two molecules. Non-covalent binding refers to a direct association between
two molecules, due to, for example, electrostatic, hydrophobic, ionic, and/or hydrogenbond
interactions, including interactions such as salt bridges and water bridges. Noncovalent
binding interactions are generally characterized by a dissociation constant (KD
) of less than 10 6 M, less than 10 7 M, less than 10 8 M, less than 10 9 M, less than 10 10
M, less than 10 11 M, less than 10 12 M, less than 10 13 M, less than 10 14 M, or less than
10 15 M. "Affinity" refers to the strength of non-covalent binding, increased binding
affinity being correlated with a lower KD. "Specific binding" generally refers to
binding with an affinity of at least about 10 7 M or greater, e.g., 5x 10 7 M, 10 8 M, 5
x 10 8 M, 10 9 M, and greater. "Non-specific binding" generally refers to binding (e.g.,
the binding of a ligand to a moiety other than its designated binding site or receptor)
with an affinity of less than about 10 7 M (e.g., binding with an affinity of 10 6 M, 10 5
M, 104 M). However, in some contexts, e.g., binding between a TCR and a peptide/
MHC complex, "specific binding" can be in the range of from 1 ~tM to 100 ~tM, or
from 100 ~tM to 1 mM. "Covalent binding" or "covalent bond," as used herein, refers
to the formation of one or more covalent chemical binds between two different
molecules.
[51] The terms "treatment", "treating" and the like are used herein to generally mean
obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic
in terms of completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete cure for a disease and/or
adverse effect attributable to the disease. "Treatment" as used herein covers any
treatment of a disease or symptom in a mammal, and includes: (a) preventing the
disease or symptom from occurring in a subject which may be predisposed to acquiring
the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the
disease or symptom, i.e., arresting its development; and/or (c) relieving the disease,
i.e., causing regression of the disease. The therapeutic agent may be administered
before, during or after the onset of disease or injury. The treatment of ongoing disease,
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where the treatment stabilizes or reduces the undesirable clinical symptoms of the
patient, is of particular interest. Such treatment is desirably performed prior to
complete loss of function in the affected tissues. The subject therapy will desirably be
administered during the symptomatic stage of the disease, and in some cases after the
symptomatic stage of the disease.
[52] The terms "individual," "subject," "host," and "patient," are used interchangeably
[53]
herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy
is desired. Mammals include, e.g., humans, non-human primates, rodents (e.g., rats;
mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and
the like), etc.
[54] Before the present invention is further described, it is to be understood that this
invention is not limited to particular embodiments described, as such may, of course,
vary. It is also to be understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended claims.
[55] Where a range of values is provided, it is understood that each intervening value, to
the tenth of the unit of the lower limit unless the context clearly dictates otherwise,
between the upper and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention. The upper and lower
limits of these smaller ranges may independently be included in the smaller ranges, and
are also encompassed within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of the limits, ranges
excluding either or both of those included limits are also included in the invention.
[56] Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the present invention, the
preferred methods and materials are now described. All publications mentioned herein
are incorporated herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited.
[57] It must be noted that as used herein and in the appended claims, the singular forms
"a," "an," and "the" include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to a "T -cell modulatory multimeric
polypeptide" includes a plurality of such polypeptides and reference to "the Immunomodulatory
polypeptide" includes reference to one or more immunomodulatory
polypeptides and equivalents thereof known to those skilled in the art, and so forth. It
is further noted that the claims may be drafted to exclude any optional element. As
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such, this statement is intended to serve as antecedent basis for use of such exclusive
terminology as "solely," "only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitation.
[58] It is appreciated that certain features of the invention, which are, for clarity,
described in the context of separate embodiments, may also be provided in combination
in a single embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment, may also be provided
separately or in any suitable sub-combination. All combinations of the embodiments
pertaining to the invention are specifically embraced by the present invention and are
disclosed herein just as if each and every combination was individually and explicitly
disclosed. In addition, all sub-combinations of the various embodiments and elements
thereof are also specifically embraced by the present invention and are disclosed herein
just as if each and every such sub-combination was individually and explicitly
disclosed herein.
[59] The publications discussed herein are provided solely for their disclosure prior to the
[60]
filing date of the present application. Nothing herein is to be construed as an admission
that the present invention is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be different from the actual
publication dates which may need to be independently confirmed.
[61] Detailed Description
[62] The present disclosure provides T -cell modulatory multimeric polypeptides that
comprise an immunomodulatory polypeptide and that comprise an epitope-presenting
Wilms tumor-1 (WT-1) peptide. A TMMP is useful for modulating the activity of aT
cell, and for modulating an immune response in an individual.
[63] T-Cell Modulatory Multimeric Polypeptides
[64] The present disclosure provides aT-cell modulatory multimeric polypeptide
(TMMP) comprising: a) a first polypeptide; and b) a second polypeptide, wherein the
TMMP comprises an epitope; a first major histocompatibility complex (MHC)
polypeptide; a second MHC polypeptide; one or more immunomodulatory
polypeptides; and optionally an immunoglobulin (lg) Fe polypeptide or a non-Ig
scaffold. The present disclosure provides a TMMP, wherein the TMMP is a heterodimer
comprising: a) a first polypeptide comprising a first MHC polypeptide; and
b) a second polypeptide comprising a second MHC polypeptide, wherein the first
polypeptide or the second polypeptide comprises an epitope (e.g., a peptide that
presents an epitope); wherein the first polypeptide and/or the second polypeptide
comprises one or more immunomodulatory polypeptides that can be the same or
different; and optionally an Ig F c polypeptide or a non-Ig scaffold. A TMMP of the
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present disclosure is also referred to herein as a "multimeric polypeptide of the present
disclosure" or a "synTac." The peptide epitope present in a TMMP of the present
disclosure is a WT -1 peptide.
[65] The present disclosure provides a TMMP comprising a heterodimeric polypeptide
comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) a first MHC
polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and c) at
least one immunomodulatory polypeptide, where the first and/or the second
polypeptide comprises the at least one (i.e., one or more) immunomodulatory
polypeptide. Optionally, the first or the second polypeptide comprises an Ig Fe
polypeptide or a non-Ig scaffold. At least one of the one or more immunomodulatory
polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity
to a cognate co-immunomodulatory polypeptide compared to the affinity of a corresponding
wild-type immunomodulatory polypeptide for the cognate coimmunomodulatory
polypeptide. The epitope present in a TMMP of the present
disclosure binds to a T -cell receptor (TCR) on a T cell with an affinity of at least 100
~LM (e.g., at least 10 ~LM, at least 1 ~LM, at least 100 nM, at least 10 nM, or at least 1
nM). A TMMP of the present disclosure binds to a first T cell with an affinity that is at
least 25% higher than the affinity with which the TMMP binds a second T cell, where
the first T cell expresses on its surface the cognate co-immunomodulatory polypeptide
and a TCR that binds the epitope with an affinity of at least 100 ~LM, and where the
second T cell expresses on its surface the cognate co-immunomodulatory polypeptide
but does not express on its surface a TCR that binds the epitope with an affinity of at
least 100 ~LM (e.g., at least 10 ~LM, at least 1 ~LM, at least 100 nM, at least 10 nM, or at
least 1 nM). In some cases, the peptide epitope present in a TMMP of the present
disclosure is a WT -1 peptide.
[66] The present disclosure provides a TMMP, wherein the TMMP is:
[67] A) a heterodimer comprising: a) a first polypeptide comprising a first MHC
polypeptide; and b) a second polypeptide comprising a second MHC polypeptide,
wherein the first polypeptide or the second polypeptide comprises an epitope (e.g., a
peptide that presents an epitope to aT cell); wherein the first polypeptide and/or the
second polypeptide comprises one or more immunomodulatory polypeptides that can
be the same or different, and wherein at least one of the one or more immunomodulatory
polypeptides may be a wild-type immunomodulatory polypeptide or
a variant of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory
polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of
the corresponding wild-type immunomodulatory polypeptide; and wherein the first
polypeptide or the second polypeptide optionally comprises an Ig Fe polypeptide or a
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non-Ig scaffold; or
[68] B) a heterodimer comprising: a) a first polypeptide comprising a first MHC
polypeptide; and b) a second polypeptide comprising a second MHC polypeptide,
wherein the first polypeptide or the second polypeptide comprises an epitope; wherein
the first polypeptide and/or the second polypeptide comprises one or more immunomodulatory
polypeptides that can be the same or different,
[69] wherein at least one of the one or more immunomodulatory polypeptides is a variant
of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory
polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of
the corresponding wild-type immunomodulatory polypeptide,
[70] wherein at least one of the one or more immunomodulatory domains is a variant immunomodulatory
polypeptide that exhibits reduced affinity to a cognate coimmunomodulatory
polypeptide compared to the affinity of a corresponding wild-type
immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide,
and wherein the epitope binds to a TCR on aT cell with an affinity of at least 10 7 M,
such that: i) the TMMP polypeptide binds to a first T cell with an affinity that is at
least 25% higher than the affinity with which the TMMP binds a second T cell,
wherein the first T cell expresses on its surface the cognate co-immunomodulatory
polypeptide and a TCR that binds the epitope with an affinity of at least 10 7 M, and
wherein the second T cell expresses on its surface the cognate co-immunomodulatory
polypeptide but does not express on its surface a TCR that binds the epitope with an
affinity of at least 10 7 M; and/or ii) the ratio of the binding affinity of a control
TMMP, wherein the control comprises a wild-type immunomodulatory polypeptide, to
a cognate co-immunomodulatory polypeptide to the binding affinity of the TMMP
comprising a variant of the wild-type immunomodulatory polypeptide to the cognate
co-immunomodulatory polypeptide, when measured by bio-layer interferometry, is in a
range of from 1.5:1 to 106:1; and wherein the variant immunomodulatory polypeptide
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid
substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory
polypeptide; and
[71] wherein the first polypeptide or the second polypeptide optionally comprises an Ig Fe
polypeptide or a non-Ig scaffold; or
[72] C) a heterodimer comprising: a) a first polypeptide comprising, in order from Nterminus
to C-terminus: i) an epitope; ii) a first MHC polypeptide; and b) a second
polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC
polypeptide; and ii) optionally an immunoglobulin (lg) Fe polypeptide or a non-Ig
scaffold, wherein the TMMP comprises one or more immunomodulatory domains that
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can be the same or different, wherein at least one of the one or more Immunomodulatory
domain is: A) at the C-terminus of the first polypeptide; B) at the Nterminus
of the second polypeptide; C) at the C-terminus of the second polypeptide; or
D) at the C-terminus of the first polypeptide and at theN-terminus of the second
polypeptide, and wherein at least one of the one or more immunomodulatory domains
may be a wild-type immunomodulatory polypeptide or a variant of a wild-type immunomodulatory
polypeptide, wherein the variant immunomodulatory polypeptide
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid
substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory
polypeptide; and
[73] optionally wherein at least one of the one or more immunomodulatory domains is a
variant immunomodulatory polypeptide that exhibits reduced affinity to a cognate coimmunomodulatory
polypeptide compared to the affinity of a corresponding wild-type
immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide,
and wherein the epitope binds to a TCR on aT cell with an affinity of at least 10 7 M,
such that: i) the TMMP binds to a first T cell with an affinity that is at least 25% higher
than the affinity with which the TMMP binds a second T cell, wherein the first T cell
expresses on its surface the cognate co-immunomodulatory polypeptide and a TCR
that binds the epitope with an affinity of at least 10 7 M, and wherein the second T cell
expresses on its surface the cognate co-immunomodulatory polypeptide but does not
express on its surface a TCR that binds the epitope with an affinity of at least 10 7 M;
and/or ii) the ratio of the binding affinity of a control TMMP, wherein the control
comprises a wild-type immunomodulatory polypeptide, to a cognate coimmunomodulatory
polypeptide to the binding affinity of the TMMP comprising a
variant of the wild-type immunomodulatory polypeptide to the cognate coimmunomodulatory
polypeptide, when measured by bio-layer interferometry, is in a
range of from 1.5:1 to 106:1; and wherein the variant immunomodulatory polypeptide
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid
substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory
polypeptide. The peptide epitope present in a TMMP of the present
disclosure is a WT -1 peptide.
[74] The present disclosure provides a TMMP comprising: a) a first polypeptide
comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first MHC
polypeptide; and b) a second polypeptide comprising, in order from N-terminus to Cterminus:
i) a second MHC polypeptide; and ii) optionally an Ig Fe polypeptide or a
non-Ig scaffold. A TMMP of the present disclosure comprises one or more immunomodulatory
polypeptides, wherein at least one of the one or more immunomodulatory
polypeptides is: A) at the C-terminus of the first polypeptide; B) at
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theN-terminus of the second polypeptide; C) at the C-terminus of the second
polypeptide; or D) at the C-terminus of the first polypeptide and at theN-terminus of
the second polypeptide. At least one of the one or more immunomodulatory
polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity
to a cognate co-immunomodulatory polypeptide compared to the affinity of a corresponding
wild-type immunomodulatory polypeptide for the cognate coimmunomodulatory
polypeptide. The epitope present in a TMMP of the present
disclosure binds to a T -cell receptor (TCR) on a T cell with an affinity of at least 100
~LM (e.g., at least 10 ~LM, at least 1 ~LM, at least 100 nM, at least 10 nM, or at least 1
nM). A TMMP of the present disclosure binds to a first T cell with an affinity that is at
least 25% higher than the affinity with which the TMMP binds a second T cell, where
the first T cell expresses on its surface the cognate co-immunomodulatory polypeptide
and a TCR that binds the epitope with an affinity of at least 100 ~LM, and where the
second T cell expresses on its surface the cognate co-immunomodulatory polypeptide
but does not express on its surface a TCR that binds the epitope with an affinity of at
least 100 ~LM (e.g., at least 10 ~LM, at least 1 ~LM, at least 100 nM, at least 10 nM, or at
least 1 nM).
[75] In some cases, the epitope present in a TMMP of the present disclosure binds to a
TCR on a T cell with an affinity of from about 104 M to about 5 x 104 M, from about
5 x 104 M to about 10 5 M, from about 10 5M to 5 x 10 5 M, from about 5 x 10 5M to
10 6 M, from about 10 6 M to about 5 x 10 6 M, from about 5 x 10 6 M to about 10 7 M,
from about 10 7 M to about 5 x 10 7 M, from about 5 x 10 7 M to about 10 8 M, or from
about 10 8M to about 10 9 M. Expressed another way, in some cases, the epitope
present in a TMMP of the present disclosure binds to a TCR on a T cell with an
affinity of from about 1 nM to about 5 nM, from about 5 nM to about 10 nM, from
about 10 nM to about 50 nM, from about 50 nM to about 100 nM, from about 0.1 ~LM
to about 0.5 ~LM, from about 0.5 ~LM to about 1 ~LM, from about 1 ~LM to about 5 ~LM,
from about 5 ~LM to about 10 ~LM, from about 10 ~LM to about 25 ~LM, from about 25
~LM to about 50 ~LM, from about 50 ~LM to about 75 ~LM, from about 75 ~LM to about
100 ~LM.
[76] An immunomodulatory polypeptide present in a TMMP of the present disclosure
binds to its cognate co-immunomodulatory polypeptide with an affinity that it at least
10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at
least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less,
at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80%
less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less, than
the affinity of a corresponding wild-type immunomodulatory polypeptide for the
cognate co-immunomodulatory polypeptide.
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[77] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the
present disclosure has a binding affinity for a cognate co-immunomodulatory
polypeptide that is from 1 nM to 100 nM, or from 100 nM to 100 ~tM. For example, in
some cases, a variant immunomodulatory polypeptide present in a TMMP of the
present disclosure has a binding affinity for a cognate co-immunomodulatory
polypeptide that is from about 100 nM to 150 nM, from about 150 nM to about 200
nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from
about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400
nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to
about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900
nM, from about 900 nM to about 1 ~tM, to about 1 ~tM to about 5 ~tM, from about 5
~tM to about 10 ~tM, from about 10 ~tM to about 15 ~tM, from about 15 ~tM to about 20
~tM, from about 20 ~tM to about 25 ~tM, from about 25 ~tM to about 50 ~tM, from about
50 ~tM to about 75 ~tM, or from about 75 ~tM to about 100 ~tM. In some cases, a variant
immunomodulatory polypeptide present in a TMMP of the present disclosure has a
binding affinity for a cognate co-immunomodulatory polypeptide that is from about 1
nM to about 5 nM, from about 5 nM to about 10 nM, from about 10 nM to about 50
nM, from about 50 nM to about 100 nM.
[78] The combination of the reduced affinity of the immunomodulatory polypeptide for
its cognate co-immunomodulatory polypeptide, and the affinity of the epitope for a
TCR, provides for enhanced selectivity of a TMMP of the present disclosure. For
example, a TMMP of the present disclosure binds selectively to a first T cell that
displays both: i) a TCR specific for the epitope present in the TMMP; and ii) a coimmunomodulatory
polypeptide that binds to the immunomodulatory polypeptide
present in the TMMP, compared to binding to a second T cell that displays: i) a TCR
specific for an epitope other than the epitope present in the TMMP; and ii) a coimmunomodulatory
polypeptide that binds to the immunomodulatory polypeptide
present in the TMMP. For example, a TMMP of the present disclosure binds to the
first T cell with an affinity that is at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, at least
15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or more
than 100-fold, higher than the affinity to which it binds the second T cell.
[79] In some cases, a TMMP of the present disclosure, when administered to an Individual
in need thereof, induces both an epitope-specific T cell response and an
epitope non-specific T cell response. In other words, in some cases, a TMMP of the
present disclosure, when administered to an individual in need thereof, induces an
epitope-specific T cell response by modulating the activity of a first T cell that displays
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both: i) a TCR specific for the epitope present in the TMMP; ii) a coimmunomodulatory
polypeptide that binds to the immunomodulatory polypeptide
present in the TMMP; and induces an epitope non-specific T cell response by
modulating the activity of a second T cell that displays: i) a TCR specific for an
epitope other than the epitope present in the TMMP; and ii) a co-immunomodulatory
polypeptide that binds to the immunomodulatory polypeptide present in the TMMP.
The ratio of the epitope-specific T cell response to the epitope-non-specific T cell
response is at least 2: 1, at least 5: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least
25: 1, at least 50: 1, or at least 100: 1. The ratio of the epitope-specific T cell response to
the epitope-non-specific T cell response is from about 2:1 to about 5:1, from about 5:1
to about 10:1, from about 10:1 to about 15:1, from about 15:1 to about 20:1, from
about 20: 1 to about 25: 1, from about 25: 1 to about 50: 1, or from about 50: 1 to about
100:1, or more than 100:1. "Modulating the activity" of aT cell can include one or
more of: i) activating a cytotoxic (e.g., CDS+) T cell; ii) inducing cytotoxic activity of a
cytotoxic (e.g., CDS+) T cell; iii) inducing production and release of a cytotoxin (e.g., a
perforin; a granzyme; a granulysin) by a cytotoxic (e.g., CDS+) T cell; iv) inhibiting
activity of an autoreactive T cell; and the like.
[SO] The combination of the reduced affinity of the immunomodulatory polypeptide for
its cognate co-immunomodulatory polypeptide, and the affinity of the epitope for a
TCR, provides for enhanced selectivity of a TMMP of the present disclosure. Thus, for
example, a TMMP of the present disclosure binds with higher avidity to a first T cell
that displays both: i) a TCR specific for the epitope present in the TMMP; and ii) a coimmunomodulatory
polypeptide that binds to the immunomodulatory polypeptide
present in the TMMP, compared to the avidity to which it binds to a second T cell that
displays: i) a TCR specific for an epitope other than the epitope present in the TMMP;
and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory
polypeptide present in the TMMP.
[Sl] Binding affinity between an immunomodulatory polypeptide and its cognate coimmunomodulatory
polypeptide can be determined by bio-layer interferometry (BLI)
using purified immunomodulatory polypeptide and purified cognate coimmunomodulatory
polypeptide. Binding affinity between a TMMP and its cognate
co-immunomodulatory polypeptide can be determined by BLI using purified TMMP
and the cognate co-immunomodulatory polypeptide. BLI methods are well known to
those skilled in the art. See, e.g., Ladet al. (2015) J. Biomol. Screen. 20(4):49S-507;
and Shah and Duncan (2014) J. Vis. Exp. 1S:e513S3.
[S2] A BLI assay can be carried out using an Octet RED 96 (Pal ForteBio) instrument, or
a similar instrument, as follows. A TMMP (e.g., a TMMP of the present disclosure; a
control TMMP (where a control TMMP comprises a wild-type immunomodulatory
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polypeptide)) is immobilized onto an insoluble support (a "biosensor"). The Immobilized
TMMP is the "target." Immobilization can be effected by immobilizing a
capture antibody onto the insoluble support, where the capture antibody immobilizes
the TMMP. For example, immobilization can be effected by immobilizing anti-Fe
(e.g., anti-human IgG Fe) antibodies onto the insoluble support, where the immobilized
anti-Fe antibodies bind to and immobilize the TMMP (where the TMMP comprises an
IgFc polypeptide). A co-immunomodulatory polypeptide is applied, at several different
concentrations, to the immobilized TMMP, and the instrument's response recorded.
Assays are conducted in a liquid medium comprising 25mM HEPES pH 6.8, 5%
poly( ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02%
Tween 20 nonionic detergent. Binding of the co-immunomodulatory polypeptide to the
immobilized TMMP is conducted at 30°C. As a positive control for binding affinity, an
anti-MHC Class I monoclonal antibody can be used. For example, anti-HLA Class I
monoclonal antibody W6/32 (American Type Culture Collection No. HB-95; Parham
et al. (1979) J. Immunol. 123:342), which has a KD of 7 nM, can be used. A standard
curve can be generated using serial dilutions of the anti-MHC Class I monoclonal
antibody. The co-immunomodulatory polypeptide, or the anti-MHC Class I mAb, is
the "analyte." BLI analyzes the interference pattern of white light reflected from two
surfaces: i) from the immobilized polypeptide ("target"); and ii) an internal reference
layer. A change in the number of molecules ("analyte"; e.g., co-immunomodulatory
polypeptide; anti-HLA antibody) bound to the biosensor tip causes a shift in the interference
pattern; this shift in interference pattern can be measured in real time. The
two kinetic terms that describe the affinity of the target/analyte interaction are the association
constant (ka) and dissociation constant (kd). The ratio of these two terms (kia)
gives rise to the affinity constant KD.
[83] The BLI assay is carried out in a multi-well plate. To run the assay, the plate layout is
defined, the assay steps are defined, and biosensors are assigned in Octet Data Acquisition
software. The biosensor assembly is hydrated. The hydrated biosensor
assembly and the assay plate are equilibrated for 10 minutes on the Octet instrument.
Once the data are acquired, the acquired data are loaded into the Octet Data Analysis
software. The data are processed in the Processing window by specifying method for
reference subtraction, y-axis alignment, inter-step correction, and Savitzky-Golay
filtering. Data are analyzed in the Analysis window by specifying steps to analyze
(Association and Dissociation), selecting curve fit model ( 1:1 ), fitting method (global),
and window of interest (in seconds). The quality of fit is evaluated. KD values for each
data trace (analyte concentration) can be averaged if within a 3-fold range. KD error
values should be within one order of magnitude of the affinity constant values; R2
values should be above 0.95. See, e.g., Abdiche et al. (2008) J. Anal. Biochem.
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377:209.
[84] Unless otherwise stated herein, the affinity of a TMMP of the present disclosure for a
cognate co-immunomodulatory polypeptide, or the affinity of a control TMMP (where
a control TMMP comprises a wild-type immunomodulatory polypeptide) for a cognate
co-immunomodulatory polypeptide, is determined using BLI, as described above.
[85] In some cases, the ratio of: i) the binding affinity of a control TMMP (where the
control comprises a wild-type immunomodulatory polypeptide) to a cognate coimmunomodulatory
polypeptide to ii) the binding affinity of a TMMP of the present
disclosure comprising a variant of the wild-type immunomodulatory polypeptide to the
cognate co-immunomodulatory polypeptide, when measured by BLI (as described
above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least
20: 1, at least 25: 1, at least 50: 1, at least 100: 1, at least 500: 1, at least 102: 1, at least 5 x
102:1, at least 103:1, at least 5 x 103:1, at least 104:1, at least 105:1, or at least 106:1. In
some cases, the ratio of: i) the binding affinity of a control TMMP (where the control
comprises a wild-type immunomodulatory polypeptide) to a cognate coimmunomodulatory
polypeptide to ii) the binding affinity of a TMMP of the present
disclosure comprising a variant of the wild-type immunomodulatory polypeptide to the
cognate co-immunomodulatory polypeptide, when measured by BLI, is in a range of
from 1.5:1 to 106:1, e.g., from 1.5:1 to 10:1, from 10:1 to 50:1, from 50:1 to 102:1,
from 102:1 to 103:1, from103:1 to 104:1, from 104:1 to 105:1, or from 105:1 to 106:1.
[86] As an example, where a control TMMP comprises a wild-type IL-2 polypeptide, and
where a TMMP of the present disclosure comprises a variant IL-2 polypeptide
(comprising from 1 to 10 amino acid substitutions relative to the amino acid sequence
of the wild-type IL-2 polypeptide) as the immunomodulatory polypeptide, the ratio of:
i) the binding affinity of the control TMMP to an IL-2 receptor (i.e., the cognate coimmunomodulatory
polypeptide) to ii) the binding affinity of the TMMP of the present
disclosure to the IL-2 receptor, when measured by BLI, is at least 1.5:1, at least 2:1, at
least 5: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 50: 1, at least
100:1, at least 500:1, at least 102:1, at least 5 x 102: 1, at least 103:1, at least 5 x 103:1, at
least 104:1, at least 105:1, or at least 106:1. In some cases, where a control TMMP
comprises a wild-type IL-2 polypeptide, and where a TMMP of the present disclosure
comprises a variant IL-2 polypeptide (comprising from 1 to 10 amino acid substitutions
relative to the amino acid sequence of the wild-type IL-2 polypeptide) as the
immunomodulatory polypeptide, the ratio of: i) the binding affinity of the control
TMMP to an IL-2 receptor (i.e., the cognate co-immunomodulatory polypeptide) to ii)
the binding affinity of the TMMP of the present disclosure to the IL-2 receptor, when
measured by BLI, is in a range of from 1.5:1 to 106:1, e.g., from 1.5:1 to 10:1, from
10:1 to 50:1, from 50:1 to 102:1, from 102:1 to 103:1, from103:1 to 104:1, from 104:1 to
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105:1, or from 105:1 to 106:1.
[87] As another example, where a control TMMP comprises a wild-type PD-L1
polypeptide, and where a TMMP of the present disclosure comprises a variant PD-L1
polypeptide (comprising from 1 to 10 amino acid substitutions relative to the amino
acid sequence of the wild-type PD-L1 polypeptide) as the immunomodulatory
polypeptide, the ratio of: i) the binding affinity of the control TMMP to a PD-1
polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding
affinity of the TMMP of the present disclosure to the PD-1 polypeptide, when
measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at
least 20: 1, at least 25: 1, at least 50: 1, at least 100: 1, at least 500: 1, at least 102: 1, at
least 5 x 102:1, at least 103:1, at least 5 x 103:1, at least 104:1, at least 105:1, or at least
106:1.
[88] As another example, where a control TMMP comprises a wild-type CD80
polypeptide, and where a TMMP of the present disclosure comprises a variant CD80
polypeptide (comprising from 1 to 10 amino acid substitutions relative to the amino
acid sequence of the wild-type CD80 polypeptide) as the immunomodulatory
polypeptide, the ratio of: i) the binding affinity of the control TMMP to a CTLA4
polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding
affinity of the TMMP of the present disclosure to the CTLA4 polypeptide, when
measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at
least 20: 1, at least 25: 1, at least 50: 1, at least 100: 1, at least 500: 1, at least 102: 1, at
least 5 x 102:1, at least 103:1, at least 5 x 103:1, at least 104:1, at least 105:1, or at least
106:1.
[89] As another example, where a control TMMP comprises a wild-type CD80
polypeptide, and where a TMMP of the present disclosure comprises a variant CD80
polypeptide (comprising from 1 to 10 amino acid substitutions relative to the amino
acid sequence of the wild-type CD80 polypeptide) as the immunomodulatory
polypeptide, the ratio of: i) the binding affinity of the control TMMP to a CD28
polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding
affinity of the TMMP of the present disclosure to the CD28 polypeptide, when
measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at
least 20: 1, at least 25: 1, at least 50: 1, at least 100: 1, at least 500: 1, at least 102: 1, at
least 5 x 102:1, at least 103:1, at least 5 x 103:1, at least 104:1, at least 105:1, or at least
106:1.
[90] As another example, where a control TMMP comprises a wild-type 4-1BBL
polypeptide, and where a TMMP of the present disclosure comprises a variant 4-1BBL
polypeptide (comprising from 1 to 10 amino acid substitutions relative to the amino
acid sequence of the wild-type 4-1BBL polypeptide) as the immunomodulatory
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polypeptide, the ratio of: i) the binding affinity of the control TMMP to a 4-lBB
polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding
affinity of the TMMP of the present disclosure to the 4-lBB polypeptide, when
measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at
least 20: 1, at least 25: 1, at least 50: 1, at least 100: 1, at least 500: 1, at least 102: 1, at
least 5 x 102:1, at least 103:1, at least 5 x 103:1, at least 104:1, at least 105:1, or at least
106:1.
[91] As another example, where a control TMMP comprises a wild-type CD86
polypeptide, and where a TMMP of the present disclosure comprises a variant CD86
polypeptide (comprising from 1 to 10 amino acid substitutions relative to the amino
acid sequence of the wild-type CD86 polypeptide) as the immunomodulatory
polypeptide, the ratio of: i) the binding affinity of the control TMMP to a CD28
polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding
affinity of the TMMP of the present disclosure to the CD28 polypeptide, when
measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at
least 20: 1, at least 25: 1, at least 50: 1, at least 100: 1, at least 500: 1, at least 102: 1, at
least 5 x 102:1, at least 103:1, at least 5 x 103:1, at least 104:1, at least 105:1, or at least
106:1.
[92] Binding affinity of a TMMP of the present disclosure to a target T cell can be
measured in the following manner: A) contacting a TMMP of the present disclosure
with a target T -cell expressing on its surface: i) a cognate co-immunomodulatory
polypeptide that binds the parental wild-type immunomodulatory polypeptide; and ii) a
T -cell receptor that binds to the epitope, where the TMMP comprises an epitope tag,
such that the TMMP binds to the target T-cell; B) contacting the target T-cell-bound
TMMP with a fluorescently labeled binding agent (e.g., a fluorescently labeled
antibody) that binds to the epitope tag, generating a TMMP/target T-cell/binding agent
complex; C) measuring the mean fluorescence intensity (MFI) of the TMMP/target Tcell/
binding agent complex using flow cytometry. The epitope tag can be, e.g., a FLAG
tag, a hemagglutinin tag, a c-myc tag, a poly(histidine) tag, etc. The MFI measured
over a range of concentrations of the TMMP library member provides a measure of the
affinity. The MFI measured over a range of concentrations of the TMMP library
member provides a half maximal effective concentration (EC50) of the TMMP. In some
cases, the EC50 of a TMMP of the present disclosure for a target T cell is in the nM
range; and the EC50 of the TMMP for a control T cell (where a control T cell expresses
on its surface: i) a cognate co-immunomodulatory polypeptide that binds the parental
wild-type immunomodulatory polypeptide; and ii) a T -cell receptor that does not bind
to the epitope present in the TMMP) is in the ~tM range. In some cases, the ratio of the
EC50 of a TMMP of the present disclosure for a control T cell to the EC50 of the TMMP
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for a target T cell is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at
least 20: 1, at least 25: 1, at least 50: 1, at least 100: 1, at least 500: 1, at least 102: 1, at
least 5 x 102:1, at least 103:1, at least 5 x 103:1, at least 104:1, at lease 105:1, or at least
106: 1. The ratio of the EC50 of a TMMP of the present disclosure for a control T cell to
the EC50 of the TMMP for a target T cell is an expression of the selectivity of the
TMMP.
[93] In some cases, when measured as described in the preceding paragraph, a TMMP of
the present disclosure exhibits selective binding to target T -cell, compared to binding
of the TMMP library member to a control T cell that comprises: i) the cognate coimmunomodulatory
polypeptide that binds the parental wild-type immunomodulatory
polypeptide; and ii) aT-cell receptor that binds to an epitope other than the epitope
present in the TMMP library member.
[94] Dimerized TMMPs
[95] A TMMP of the present disclosure can be dimerized; i.e., the present disclosure
provides a multimeric polypeptide comprising a dimer of a TMMP of the present
disclosure. Thus, the present disclosure provides a TMMP comprising: A) a first heterodimer
comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) a
first major histocompatibility complex (MHC) polypeptide; and b) a second
polypeptide comprising: i) a second MHC polypeptide, wherein the first heterodimer
comprises one or more immunomodulatory polypeptides; and B) a second heterodimer
comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) a first MHC
polypeptide; and b) a second polypeptide comprising: i) a second MHC polypeptide,
wherein the second heterodimer comprises one or more immunomodulatory
polypeptides, and wherein the first heterodimer and the second heterodimer are covalently
linked to one another. In some cases, the two TMMPs are identical to one
another in amino acid sequence. In some cases, the first heterodimer and the second
heterodimer are covalently linked to one another via a C-terminal region of the second
polypeptide of the first heterodimer and a C-terminal region of the second polypeptide
of the second heterodimer. In some cases, first heterodimer and the second heterodimer
are covalently linked to one another via the C-terminal amino acid of the second
polypeptide of the first heterodimer and the C-terminal region of the second
polypeptide of the second heterodimer; for example, in some cases, the C-terminal
amino acid of the second polypeptide of the first heterodimer and the C-terminal
region of the second polypeptide of the second heterodimer are linked to one another,
either directly or via a linker. The linker can be a peptide linker. The peptide linker can
have a length of from 1 amino acid to 200 amino acids (e.g., from 1 amino acid (aa) to
5 aa, from 5 aa to 10 aa, from 10 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa,
from 100 aa to 150 aa, or from 150 aa to 200 aa). In some cases, the peptide epitope of
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the first heterodimer and the peptide epitope of the second heterodimer comprise the
same amino acid sequence. In some cases, the first MHC polypeptide of the first and
the second heterodimer is an MHC Class I ~2-microglobulin, and wherein the second
MHC polypeptide of the first and the second heterodimer is an MHC Class I heavy
chain. In some cases, the immunomodulatory polypeptide of the first heterodimer and
the immunomodulatory polypeptide of the second heterodimer comprise the same
amino acid sequence. In some cases, the immunomodulatory polypeptide of the first
heterodimer and the immunomodulatory polypeptide of the second heterodimer are
variant immunomodulatory polypeptides that comprise from 1 to 10 amino acid substitutions
compared to a corresponding parental wild-type immunomodulatory
polypeptide, and wherein the from 1 to 10 amino acid substitutions result in reduced
affinity binding of the variant immunomodulatory polypeptide to a cognate coimmunomodulatory
polypeptide. In some cases, the immunomodulatory polypeptide of
the first heterodimer and the immunomodulatory polypeptide of the second heterodimer
are each independently selected from the group consisting of IL-2, 4-1BBL,
PD-L1, CD80, CD86, ICOS-L, OX-40L, FasL, JAG1 (CD339), TGF~, CD70, and
ICAM. Examples, of suitable MHC polypeptides, immunomodulatory polypeptides,
and peptide epitopes are described below.
[96] MHC polypeptides
[97] As noted above, a TMMP of the present disclosure includes MHC polypeptides. For
the purposes of the instant disclosure, the term "major histocompatibility complex
(MHC) polypeptides" is meant to include MHC polypeptides of various species,
including human MHC (also referred to as human leukocyte antigen (HLA))
polypeptides, rodent (e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides
of other mammalian species (e.g., lagomorphs, non-human primates, canines, felines,
ungulates (e.g., equines, bovines, ovines, caprines, etc.), and the like. The term "MHC
polypeptide" is meant to include Class I MHC polypeptides (e.g., ~-2 microglobulin
and MHC class I heavy chain).
[98] In some cases, the first MHC polypeptide is an MHC Class I ~2M (~2M)
polypeptide, and the second MHC polypeptide is an MHC Class I heavy chain (H
chain) ("MHC-H")). In other instances, the first MHC polypeptide is an MHC Class I
heavy chain polypeptide; and the second MHC polypeptide is a ~2M polypeptide. In
some cases, both the ~2M and MHC-H chain are of human origin; i.e., the MHC-H
chain is an HLA heavy chain, or a variant thereof. Unless expressly stated otherwise, a
TMMP of the present disclosure does not include membrane anchoring domains
(transmembrane regions) of an MHC Class I heavy chain, or a part of MHC Class I
heavy chain sufficient to anchor the resulting TMMP to a cell (e.g., eukaryotic cell
such as a mammalian cell) in which it is expressed. In some cases, the MHC Class I
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heavy chain present in a TMMP of the present disclosure does not include a signal
peptide, a transmembrane domain, or an intracellular domain (cytoplasmic tail) associated
with a native MHC Class I heavy chain. Thus, e.g., in some cases, the MHC
Class I heavy chain present in a TMMP of the present disclosure includes only the a1,
a2, and a3 domains of an MHC Class I heavy chain. In some cases, the MHC Class I
heavy chain present in a TMMP of the present disclosure has a length of from about
270 amino acids (aa) to about 290 aa. In some cases, the MHC Class I heavy chain
present in a TMMP of the present disclosure has a length of 270 aa, 271 aa, 272 aa,
273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa, 280 aa, 281 aa, 282 aa, 283 aa,
284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290 aa.
[99] In some cases, an MHC polypeptide of a TMMP is a human MHC polypeptide,
where human MHC polypeptides are also referred to as "human leukocyte antigen"
("HLA") polypeptides. In some cases, an MHC polypeptide of a TMMP is a Class I
HLA polypeptide, e.g., a ~2-microglobulin polypeptide, or a Class I HLA heavy chain
polypeptide.
[100] MHC Class I heavy chains
[101] In some cases, an MHC Class I heavy chain polypeptide present in a TMMP of the
[102]
present disclosure comprises an amino acid sequence having at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%,
amino acid sequence identity to all or part (e.g., 50, 75, 100, 150, 200, or 250
contiguous amino acids) of the amino acid sequence of the human HLA heavy chain
polypeptides depicted in FIG. 6. In some cases, the MHC Class I heavy chain has a
length of 270 aa, 271 aa, 272 aa, 273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa,
280 aa, 281 aa, 282 aa, 283 aa, 284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290
aa. In some cases, an MHC Class I heavy chain polypeptide present in a TMMP of the
present disclosure comprises 1-30, 1-5, 5-10, 10-15, 15-20, 20-25 or 25-30 amino acid
insertions, deletions, and/or substitutions (in addition to those locations indicated as
being variable in the heavy chain consensus sequences) of the amino acid sequences
depicted in FIG. 6. In some cases, the MHC Class I heavy chain does not include transmembrane
or cytoplasmic domains. As an example, a MHC Class I heavy chain
polypeptide of a TMMP of the present disclosure can comprise an amino acid
sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 25-300
or amino acids 25-299 (lacking all, or substantially all, of the leader, transmembrane
and cytoplasmic sequence) or amino acids 25-365 (lacking the leader) of a human
HLA-A heavy chain polypeptides depicted in FIG. 6.
[103] HLA-A
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[104] In some cases, a TMMP of the present disclosure comprises an HLA-A heavy chain
polypeptide. The HLA-A heavy chain peptide sequences, or portions thereof, that may
be that may be incorporated into a TMMP of the present disclosure include an HLA
A *2402 allele heavy chain, without all, or substantially all, of the leader, transmembrane
and cytoplasmic sequences. Any of those alleles may comprise a mutation
at one or more of positions 84, 139 and/or 236 (as shown in FIG. 6) selected from: a
tyrosine to alanine at position 84 (Y84A); a tyrosine to cysteine at position 84 (Y84C);
an alanine to cysteine at position 139 (A139C); and an alanine to cysteine substitution
at position 236 (A236C). In addition, HLA-A sequence having at least 75% (e.g., at
least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100%
amino acid sequence identity to all or part (e.g., 50, 75, 100, 150, 200, or 250
contiguous amino acids) of the sequence of an HLA-A *2402 heavy chain allele may
also be employed (e.g., it may comprise 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30
amino acid insertions, deletions, and/or substitutions).
[105] In some cases, a TMMP of the present disclosure comprises an HLA-A heavy chain
polypeptide comprising the following HLA-A consensus amino acid sequence:
[ 1 06] GSHSMRYFXl TSVSRPGRGEPRFIA VGYVDDTQFVRFDSDAASQX2MEPRAP
WIEQEGPEYWDX3X4TX5X6X7KAX8SQX9X10RX11X12LX13X14X15X16Xl7
YYNQSEX18GSHTX19QX20MX21GCDVGX22DX23RFLRGYX24QX25AYDGK
DYIALX26EDLRSWTAADMAAQX27TX287X29KWEX30X31X32EAEQX33RX3
~YLX35GX36CVX37X38LRRYLENGKETLQRTDX39PKTHMTHHX40X41SDHE
ATLRCW ALX42FYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKW AX43
VVVPSGX44EQRYTCHVQHEGLPKPLTLRWEX45 (SEQ ID N0:19), wherein X1
is F, Y, S, or T; X2 is K orR; X3 is Q, G, E, orR; X4 is Nor E; X5 is R or G; X6 is N
or K; X7 isM or V; X8 isH or Q; X9 is Tor I; X10 is D or H; X11 is A, V, orE; X12
is NorD; X13 is G orR; X14 is Tor I; X15 is Lor A; X16 is R or L; X17 is G orR;
X18 is A or D; X19 is I, L, or V; X20 is I, R or M; X21 is For Y; X22 isS or P; X23
is W or G; X24 is R, H, or Q; X25 is DorY; X26 is Nor K; X27 is Tor I; X28 is K or
Q; X29 is R or H; X30 is A or T; X31 is A or V; X32 isH orR; X33 is R, L, Q, or W;
X34 is V or A; X35 is D orE; X36 is R or T; X37 is D or E; X38 is W or G; X39 is P
or A; X40 is P or A; X41is V or I; X42 isS or G; X43 is A or S; X44 is Q orE; and
X45 is P or L.
[107] HLA-A24 (HLA-A*2402)
[108] As one non-limiting example, an MHC Class I heavy chain polypeptide of a TMMP
of the present disclosure can comprise an amino acid sequence having at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or
100%, amino acid sequence identity to the following human HLA-A24 (also referred
to as HLA-A*2402) heavy chain amino acid sequence: GSHSMRYFSTSVSRPwo
2021/230638 PCT/KR2021/005913
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GRGEPRFIA VGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKV
KAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY A Y
DGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLR
RYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGE
DQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLR
WEPSSQPTVPIVGIIAGL VLLGA VITGA VV AA VMWRRNSSDRKGGSYSQAASS
DSAQGSDVSLTACKV (SEQ ID N0:20). Such an MHC Class I heavy chain may be
prominent in Asian populations, including populations of individuals of Asian descent.
In some cases, amino acid 84 is an Ala. In some cases, amino acid 84 is a Cys. In some
cases, amino acid 236 is a Cys. In some cases, amino acid 84 is an Ala and amino acid
236 is a Cys. In some cases, amino acid 84 is a Cys and amino acid 236 is a Cys.
[109] In some cases, an MHC Class I heavy chain polypeptide of a TMMP of the present
disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following human HLA-A24 (also referred to as HLA-A *2402)
heavy chain amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIA VGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIA
LRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY A YDGKDYIALKEDLRS
WTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRT
DPPKTHMTHHPISDHEATLRCW ALGFYPAEITLTWQRDGEDQTQDTELVETRP
AGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID N0:21),
where amino acid 84 is Tyr and amino acid 236 is Ala (amino acids 84 and 236 are
bold and underlined); and where the MHC Class I heavy chain has a length of about
275 amino acids.
[110] In some cases, an MHC Class I heavy chain polypeptide of a TMMP of the present
disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following human HLA-A24 (also referred to as HLA-A *2402)
heavy chain amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIA VGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIA
LRA YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY A YDGKDYIALKEDLRS
WTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRT
DPPKTHMTHHPISDHEATLRCW ALGFYPAEITLTWQRDGEDQTQDTELVETRP
AGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID N0:22),
where amino acid 84 is Ala and amino acid 236 is Ala (amino acids 84 and 236 are
bold and underlined); and where the MHC Class I heavy chain has a length of about
275 amino acids.
[111] In some cases, an MHC Class I heavy chain polypeptide of a TMMP of the present
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disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following human HLA-A24 (also referred to as HLA-A *2402)
heavy chain amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIA VGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIA
LRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY A YDGKDYIALKEDLRS
WTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRT
DPPKTHMTHHPISDHEATLRCW ALGFYPAEITLTWQRDGEDQTQDTELVETRP
CGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID N0:23),
where amino acid 84 is Tyr and amino acid 236 is Cys (amino acids 84 and 236 are
bold and underlined); and where the MHC Class I heavy chain has a length of about
275 amino acids.
[112] In some cases, an MHC Class I heavy chain polypeptide of a TMMP of the present
disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following human HLA-A24 (also referred to as HLA-A *2402)
heavy chain amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIA VGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIA
LRA YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY A YDGKDYIALKEDLRS
WTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRT
DPPKTHMTHHPISDHEATLRCW ALGFYPAEITLTWQRDGEDQTQDTELVETRP
CGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID N0:24),
where amino acid 84 is Ala and amino acid 236 is Cys (amino acids 84 and 236 are
bold and underlined); and where the MHC Class I heavy chain has a length of about
275 amino acids.
[113] In some cases, an MHC Class I heavy chain polypeptide of a TMMP of the present
disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following human HLA-A24 (also referred to as HLA-A *2402)
heavy chain amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIA VGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIA
LRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY A YDGKDYIALKEDLRS
WTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRT
DPPKTHMTHHPISDHEATLRCW ALGFYPAEITLTWQRDGEDQTQDTELVETRP
AGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID N0:25),
where amino acid 84 is Cys and amino acid 236 is Ala (amino acids 84 and 236 are
bold and underlined); and where the MHC Class I heavy chain has a length of about
275 amino acids.
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[114] In some cases, an MHC Class I heavy chain polypeptide of a TMMP of the present
disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following human HLA-A24 (also referred to as HLA-A *2402)
heavy chain amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIA VGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIA
LRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY A YDGKDYIALKEDLRS
WTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRT
DPPKTHMTHHPISDHEATLRCW ALGFYPAEITLTWQRDGEDQTQDTELVETRP
CGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID N0:26),
where amino acid 84 is Cys and amino acid 236 is Cys (amino acids 84 and 236 are
bold and underlined); and where the MHC Class I heavy chain has a length of about
275 amino acids.
[115] Beta-2 microglobulin
[116] A ~2-microglobulin (~2M) polypeptide of a TMMP of the present disclosure can be
a human ~2M polypeptide, a non-human primate ~2M polypeptide, a murine ~2M
polypeptide, and the like. In some instances, a ~2M polypeptide comprises an amino
acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a ~2M
amino acid sequence depicted in FIG. 6. In some instances, a ~2M polypeptide
comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence
identity to amino acids 21 to 119 of a ~2M amino acid sequence depicted in FIG. 6.
[117] In some cases, a suitable ~2M polypeptide comprises the following amino acid
sequence:
[118] IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE
HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM
(SEQ ID N0:32); and the HLA Class I heavy chain polypeptide comprises the
following amino acid sequence:
[119] GSHSMRYFFTSVSRPGRGEPRFIA VGYVDDTQFVRFDSDAASQRMEPRAPWI
EQEGPEYWDGETRKVKAHSQTHRVDL(aa1){C}(aa2)AGSHTVQRMYGCDVGS
DWRFLRGYHQYAYDGKDYIALKEDLRSW(aa3){C}(aa4))HKWEAAHVAEQLR
AYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSF
YPAEITLTWQRDGEDQTQDTEL( aa5)(C)( aa6)QKW AA VVVPSGQEQRYTCHVQ
HEGLPKPLTLRWEP (SEQ ID N0:27), where the cysteine residues indicated as { C}
form an disulfide bond between the a1 and a2-1 helices and the (C) residue forms a
disulfide bond with the ~2M polypeptide cysteine at position 12. In the sequence
above, "aa1" is "amino acid cluster 1" ; "aa2" is "amino acid cluster 2"; "aa3" is "amino
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acid cluster 3"; "aa4" is "amino acid cluster 4" ; "aa5" is "amino acid cluster 5"; and
"aa6" is "amino acid cluster 6"; see, e.g., FIG. 10. Each occurrence of aa1, aa2, aa3,
aa4, aa5, and aa6 is and independently selected to be 1-5 amino acid residues, wherein
the amino acid residues are i) selected independently from any naturally occurring
(e.g., encoded) amino acid or ii) any naturally occurring amino acid except proline or
glycine.
[120] In some cases, an MHC polypeptide comprises a single amino acid substitution
relative to a reference MHC polypeptide (where a reference MHC polypeptide can be a
wild-type MHC polypeptide), where the single amino acid substitution substitutes an
amino acid with a cysteine (Cys) residue. Such cysteine residues, when present in an
MHC polypeptide of a first polypeptide of a TMMP of the present disclosure, can form
a disulfide bond with a cysteine residue present in a second polypeptide chain of a
TMMP of the present disclosure.
[121] In some cases, a first MHC polypeptide in a first polypeptide of a TMMP of the
present disclosure, and/or the second MHC polypeptide in the second polypeptide of a
TMMP of the present disclosure, includes an amino acid substitution to substitute an
amino acid with a cysteine, where the substituted cysteine in the first MHC
polypeptide forms a disulfide bond with a cysteine in the second MHC polypeptide,
where a cysteine in the first MHC polypeptide forms a disulfide bond with the substituted
cysteine in the second MHC polypeptide, or where the substituted cysteine in
the first MHC polypeptide forms a disulfide bond with the substituted cysteine in the
second MHC polypeptide.

Claims
AT-cell modulatory multimeric polypeptide comprising:
at least one heterodimer comprising:
a) a first polypeptide comprising:
i) a Wilms tumor -1 (WT -1) peptide epitope having a length of from
9-25 amino acids comprising an amino acid sequence selected from the
group consisting of 302-310 (RVPGVAPTL) (SEQ ID N0:80),
302-310;V303Y (RYPGVAPTL) (SEQ ID N0:81), 126-134;Ml27Y
(RYFPNAPYL) (SEQ ID N0:82), and 417-425;W418Y
(RYPSCQKKF) (SEQ ID N0:83), and
ii) a first Class I major histocompatibility complex (MHC) polypeptide;
b) a second polypeptide comprising a second class I MHC polypeptide,
and
c) at least one activating immunomodulatory polypeptide,
wherein the first and/or the second polypeptide comprises the at least
one immunomodulatory polypeptide, and optionally wherein the first or
the second polypeptide comprises an immunoglobulin (Ig) Fe
polypeptide.
AT-cell modulatory multimeric polypeptide of claim 1, wherein at
least one of the one or more immunomodulatory polypeptides is a
variant immunomodulatory polypeptide that exhibits reduced affinity to
a cognate co-immunomodulatory polypeptide compared to the affinity
of a corresponding wild-type immunomodulatory polypeptide for the
cognate co-immunomodulatory polypeptide.
AT-cell modulatory multimeric polypeptide of claim 2, wherein the
ratio of the binding affinity of the wild-type immunomodulatory
polypeptide to a cognate co-immunomodulatory polypeptide to the
binding affinity of the variant immunomodulatory polypeptide to the
cognate co-immunomodulatory polypeptide, when measured by biolayer
interferometry, is at least 1.5:1.
AT-cell modulatory multimeric polypeptide of claim 2 or 3, wherein
the variant immunomodulatory polypeptide binds the coimmunomodulatory
polypeptide with an affinity selected from the
group consisting of from about 104 M to about 10 7 M, from about 104
M to about 10 6M, and from about 104 M to about 10 5 M.
AT-cell modulatory multimeric polypeptide of any one of claims 1-4,
wherein
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a1) the first polypeptide comprises, in order from N-terminus to Cterminus:
i) the WT -1 peptide epitope; and
ii) the first MHC polypeptide; and
b1) the second polypeptide comprises, in order from N-terminus to Cterminus:
i) the at least one immunomodulatory polypeptide;
ii) the second MHC polypeptide; and
iii) an Ig Fe polypeptide; or
a2) the first polypeptide comprises, in order from N-terminus to Cterminus:
i) the WT -1 peptide epitope; and
ii) the first MHC polypeptide; and
b2) the second polypeptide comprises, in order from N-terminus to Cterminus:
i) the second MHC polypeptide;
ii) the at least one immunomodulatory polypeptide; and
iii) an Ig Fe polypeptide; or
a3) the first polypeptide comprises, in order from N-terminus to Cterminus:
i) the WT -1 peptide epitope; and
ii) the first MHC polypeptide; and
b3) the second polypeptide comprises, in order from N-terminus to Cterminus:
i) the second MHC polypeptide;
ii) an Ig Fe polypeptide; and
iii) the at least one immunomodulatory polypeptide; or
a4) the first polypeptide comprises, in order from N-terminus to Cterminus:
i) the at least one immunomodulatory polypeptide;
ii) the WT -1 peptide epitope;
iii) the first MHC polypeptide; and
b4) the second polypeptide comprises, in order from N-terminus to Cterminus:
i) the second MHC polypeptide; and
ii) the Ig Fe polypeptide; or
a5) the first polypeptide comprises, in order from N-terminus to Cterminus:
wo 2021/230638
[Claim 6]
[Claim 7]
[Claim 8]
[Claim 9]
PCT/KR2021/005913
142
i) the WT -1 peptide epitope;
ii) the first MHC polypeptide; and
iii) the at least one immunomodulatory polypeptide; and
b5) the second polypeptide comprises, in order from N-terminus to Cterminus:
i) the second MHC polypeptide; and
ii) an immunoglobulin (Ig) Fe polypeptide.
AT-cell modulatory multimeric polypeptide of any one of claims 1-4,
wherein:
a) the first MHC polypeptide is a ~2-microglobulin polypeptide; and
the second MHC polypeptide is an MHC class I heavy chain
polypeptide; or
b) the first MHC polypeptide is an MHC class I heavy chain
polypeptide; and the second MHC polypeptide is a ~2-microglobulin
polypeptide.
AT-cell modulatory multimeric polypeptide of claim 6, wherein:
a) the first polypeptide comprises, in order from N-terminus to Cterminus:
i) the WT -1 peptide epitope; and
ii) the ~2-microglobulin polypeptide; and
b) the second polypeptide comprises, in order from N-terminus to Cterminus:
i) the at least one immunomodulatory polypeptide;
ii) the MHC class I heavy chain polypeptide; and
iii) an Ig Fe polypeptide.
AT-cell modulatory multimeric polypeptide of claim 6, wherein:
a) the first polypeptide comprises, in order from N-terminus to Cterminus:
i) the WT -1 peptide epitope; and
ii) the ~2-microglobulin polypeptide; and
b) the second polypeptide comprises, in order from N-terminus to Cterminus:
i) the MHC class I heavy chain polypeptide; and
ii) an Ig Fe polypeptide; and
iii) at least one immunomodulatory polypeptide
AT-cell modulatory multimeric polypeptide of any one of claims 1-8,
wherein the at least one immunomodulatory polypeptide is selected
from the group consisting of a cytokine, a 4-lBBL polypeptide, an
wo 2021/230638
[Claim 10]
[Claim 11]
[Claim 12]
[Claim 13]
[Claim 14]
[Claim 15]
[Claim 16]
PCT/KR2021/005913
143
ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a
CD86 polypeptide, a CD40 polypeptide, a CD70 polypeptide, and combinations
thereof.
AT-cell modulatory multimeric polypeptide of any one of claims 1-9,
wherein the at least one immunomodulatory polypeptide comprises an
IL-2 polypeptide.
AT-cell modulatory multimeric polypeptide of any one of claims 1-10,
wherein the multimeric polypeptide comprises at least two immunomodulatory
polypeptides, and wherein at least two of the immunomodulatory
polypeptides are the same, optionally wherein the 2
or more immunomodulatory polypeptides are in tandem.
AT-cell modulatory multimeric polypeptide of any one of claims 1-11,
wherein one or more of the at least one immunomodulatory polypeptide
is a variant IL-2 polypeptide that exhibits reduced affinity to an IL-2
receptor compared to the affinity of a wild-type IL-2 polypeptide for
the IL-2 receptor.
AT-cell modulatory multimeric polypeptide of claim 12, wherein the
one or more variant IL-2 polypeptides comprises: i) an Hl6A substitution
and an F42A substitution; or ii) an Hl6T substitution and an
F42A substitution.
AT-cell modulatory multimeric polypeptide of any one of claims 1-13,
wherein the first polypeptide and the second polypeptide are covalently
linked to one another, optionally wherein the covalent linkage is via a
disulfide bond.
AT-cell modulatory multimeric polypeptide of any one of claims 1-14,
wherein the first MHC polypeptide or a linker between the epitope and
the first MHC polypeptide comprises an amino acid substitution to
provide a first Cys residue, wherein the second MHC polypeptide
comprises an amino acid substitution to provide a second Cys residue,
and wherein the disulfide linkage is between the first and the second
Cys residues.
AT-cell modulatory multimeric polypeptide of any one of claims 1-15,
wherein the polypeptide comprises a disulfide bond between: i) a Cys
present in a linker between the WT -1 peptide epitope and the first
MHC class I polypeptide, wherein the first MHC class I polypeptide is
a ~2M polypeptide; and ii) a Cys residue introduced via a Y84C substitution
in the second MHC class I polypeptide, wherein the second
MHC class I polypeptide is a MHC Class I heavy chain polypeptide.
wo 2021/230638
[Claim 17]
[Claim 18]
[Claim 19]
[Claim 20]
[Claim 21]
[Claim 22]
[Claim 23]
PCT/KR2021/005913
144
AT-cell modulatory multimeric polypeptide of any one of claims 1-15,
wherein the polypeptide comprises a disulfide bond between i) a Cys
residue introduced into the first MHC class I polypeptide via an Rl2C
substitution, wherein the first MHC class I polypeptide is a ~2M
polypeptide; and ii) a Cys residue introduced into the second MHC
class I polypeptide, via an A236C substitution, wherein second MHC
class I polypeptide is an MHC Class I heavy chain polypeptide.
AT-cell modulatory multimeric polypeptide of any one of claims 1-15,
wherein the polypeptide comprises a first disulfide bond between: i) a
Cys present in a linker between the WT -1 peptide epitope and the first
MHC class I polypeptide, wherein the first MHC class I polypeptide is
a ~2M polypeptide; and ii) a Cys residue introduced via a Y84C substitution
in the second MHC class I polypeptide, wherein the second
MHC class I polypeptide is a MHC Class I heavy chain polypeptide,
and a second disulfide bond between i) a Cys residue introduced into
the ~2M polypeptide via an Rl2C substitution; and ii) a Cys residue introduced
into the MHC Class I heavy chain polypeptide via an A236C
substitution.
AT-cell modulatory multimeric polypeptide of claim 16 or claim 18,
wherein the linker between the WT -1 peptide epitope and the first
MHC is GCGGS(GGGGS)n (SEQ ID N0:33), where n is 1, 2, 3, 4, 5,
6, 7, 8, or 9.
AT-cell modulatory multimeric polypeptide of any one of claims 1-19,
wherein the WT -1 peptide epitope has a length of 9 amino acids.
AT-cell modulatory multimeric polypeptide of any one of claims 1-20,
wherein the Ig Fe polypeptide comprises one of the amino acid
sequences depicted in FIG. 4D, FIG, 4E, FIG. 4F, FIG. 4G, and FIG.
4H.
AT-cell modulatory multimeric polypeptide of any one of claims 1-21,
wherein the WT-1 peptide comprises the amino acid sequence 302-310
(RVPGVAPTL) (SEQ ID N0:80), 302-310;V303Y (RYPGVAPTL)
(SEQ ID N0:81), 126-134;Ml27Y (RYFPNAPYL) (SEQ ID N0:82),
and 417-425;W418Y (RYPSCQKKF) (SEQ ID N0:83).
AT-cell modulatory multimeric polypeptide of any one of claims 1-21,
wherein the first or the second MHC polypeptide comprises an amino
acid sequence having at least 95% amino acid sequence identity to
amino acids 25-299 of the HLA-A *2402 amino acid sequence depicted
in FIG. 6.
wo 2021/230638
[Claim 24]
[Claim 25]
[Claim 26]
[Claim 27]
[Claim 28]
[Claim 29]
[Claim 30]
[Claim 31]
[Claim 32]
PCT/KR2021/005913
145
AT-cell modulatory multimeric polypeptide of any one of claims 1-23,
wherein the first MHC polypeptide is a ~2M polypeptide, and wherein
the second MHC polypeptide comprises an amino acid sequence having
at least 95% amino acid sequence identity to an HLA-A24 polypeptide,
wherein the epitope is selected from the group consisting of: 302-310
(RVPGVAPTL) (SEQ ID N0:80), 302-310;V303Y (RYPGVAPTL)
(SEQ ID N0:81), 126-134;Ml27Y (RYFPNAPYL) (SEQ ID N0:82),
and 417-425;W418Y (RYPSCQKKF) (SEQ ID N0:83), and wherein
the Ig Fe polypeptide comprises the amino acid sequence depicted in
FIG. 4G or in FIG. 4H.
AT-cell modulatory multimeric polypeptide of claim 1, wherein:
a) the first polypeptide comprises the amino acid sequence depicted in
FIG. 13B; and
b) the second polypeptide comprises the amino acid sequence depicted
in FIG. lOB.
AT-cell modulatory multimeric polypeptide of claim 1, wherein:
a) the first polypeptide comprises the amino acid sequence depicted in
FIG. 12B; and
b) the second polypeptide comprises the amino acid sequence depicted
in FIG. lOB.
AT-cell modulatory multimeric polypeptide of any one of claims 1-26,
wherein the multimeric polypeptide comprises a first and a second heterodimer,
and wherein the first and second heterodimers are covalently
bound by one or more disulfide bonds between the Ig Fe polypeptides
of the first and second heterodimers.
A nucleic acid comprising a nucleotide sequence encoding a first or
second polypeptide according to any one of claims 1-27.
An expression vector comprising the nucleic acid of claim 28.
A method of selectively modulating the activity ofT cell specific for a
Wilms tumor-1 (WT-1) epitope, the method comprising contacting the
T cell with aT-cell modulatory multimeric polypeptide according to
any one of claims 1-27, wherein said contacting selectively modulates
the activity of the WT -1 epitope-specific T cell.
A method of treating a patient having a cancer, the method comprising
administering to the patient an effective amount of a pharmaceutical
composition comprising T -cell modulatory multimeric polypeptide
according to any one of claims 1-27.
The method of claim 31, wherein the cancer is acute myeloid leukemia,
wo 2021/230638
[Claim 33]
[Claim 34]
[Claim 35]
[Claim 36]
[Claim 37]
[Claim 38]
PCT/KR2021/005913
146
myeloma, ovarian cancer, pancreatic cancer, non-small cell lung
cancer, colorectal cancer, breast cancer, Wilms tumor, mesothelioma,
soft tissue sarcoma, neuroblastoma, or nephroblastoma.
A method of claim 31 or 32, further comprising administering one or
more checkpoint inhibitors to the individual.
A method according to claim 33, wherein the checkpoint inhibitor is an
antibody that binds to a polypeptide selected from the group consisting
of CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR,
CSFlR, JAK, PBK delta, PBK gamma, TAM, arginase, CD137,
ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA,
CD96, TIGIT, CD122, PD-1, PD-Ll, and PD-L2.
A method according to claim 34, wherein the checkpoint inhibitor is an
antibody specific for PD-1, PD-Ll, or CTLA4.
A method according to claim 34 or 35, wherein the one or more
checkpoint inhibitors is selected from the group consisting of
nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A,
MDX-1105, MEDI-4736, arelumab, ipilimumab, tremelimumab,
pidilizumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab,
PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab,
Varlilumab, Avelumab, Galiximab, AMP-514, AUNP 12, Indoximod,
NLG-919, INCB024360, KN035, and combinations thereof.
A method of modulating an immune response in an individual, the
method comprising administering to the individual an effective amount
of the T -cell modulatory multimeric polypeptide of any one of claims
1-27,
wherein said administering induces an epitope-specific T cell response
and an epitope-non-specific T cell response, and
wherein the ratio of the epitope-specific T cell response to the epitopenon-
specific T cell response is at least 2:1.
A method of delivering an immunomodulatory polypeptide selectively
to a target T cell, the method comprising contacting a mixed population
ofT cells with aT -cell modulatory multimeric polypeptide of any one
of claims 1-27, wherein the mixed population ofT cells comprises the
target T cell and non-target T cells, wherein the target T cell is specific
for the WT -1 epitope present within the T -cell modulatory multimeric
polypeptide, and wherein said contacting delivers the one or more immunomodulatory
polypeptides present within the T -cell modulatory
multimeric polypeptide to the target T cell.
wo 2021/230638
[Claim 39]
PCT/KR2021/005913
147
A method of detecting, in a mixed population ofT cells obtained from
an individual, the presence of a target T cell that binds a WT -1 epitope,
the method comprising:
a) contacting in vitro the mixed population ofT cells with the T -cell
modulatory multimeric polypeptide of any one of claims 1-27, wherein
the T-cell modulatory multimeric polypeptide comprises the WT-1
epitope; and
b) detecting activation and/or proliferation ofT cells in response to said
contacting, wherein activated and/or proliferated T cells indicates the
presence of the target T cell.