Patent Application
20240317815
A Sequence Listing is provided herewith as a Sequence Listing XML, “CUEB-145WO_SEQUENCE_LIST” created on Nov. 17, 2022 and having a size of 1,190,000 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
An adaptive immune response involves the engagement of the T cell receptor (TCR), present on the surface of a T 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 for T 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—epitope/TCR 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.
The present disclosure provides T-cell modulatory multimeric polypeptides (TMMPs) that comprise an immunomodulatory polypeptide, class I HLA polypeptides (a class I HLA heavy chain polypeptide and a β2 microglobulin polypeptide), a peptide that presents an epitope to a T-cell receptor, and a tumor-targeting polypeptide. A TMMP of the present disclosure is useful for modulating the activity of a T cell, and for modulating an immune response in an individual and for “redirecting” a patient's repertoire of antiviral T cells to attack and kill cancer cells.
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 multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
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. Furthermore, as used herein, a “polypeptide” refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to polymerase chain reaction (PCR) amplification or other recombinant DNA methods. References herein to a specific residue or residue number in a known polypeptide are understood to refer to the amino acid at that position in the wild-type polypeptide. To the extent that the sequence of the wild-type polypeptide is altered, either by addition or deletion of one or more amino acids, one of ordinary skill will understand that a reference to the specific residue or residue number will be correspondingly altered so as to refer to the same specific amino acid in the altered polypeptide, which would be understood to reside at an altered position number. For example, if an MHC class I polypeptide is altered by the addition of one amino acid at the N-terminus, then a reference to position 84 or a specific residue at position 84, will be understood to indicate the amino acids that are at position 85 on the altered polypeptide. Likewise, a reference herein to substitution of a specific amino acid at a specific position, e.g., Y84, is understood to refer to a substitution of an amino acid for the amino acid at position 84 in the wild-type polypeptide. A Y84C substitution is thus understood to be a substitution of Cys residue for the Tyr residue that is present in the wild-type sequence. If, e.g., the wild-type polypeptide is altered to change the amino acid at position 84 from its wild-type amino acid to an alternate amino acid, then the substitution for the amino acid at position 84 will be understood to refer to the substitution for the alternate amino acid. If in such case the polypeptide is also altered by the addition or deletion of one or more amino acids, then the reference to the substitution will be understood to refer to the substitution for the alternate amino acid at the altered position number. A reference to a “non-naturally occurring Cys residue” in a polypeptide, e.g., an MHC class I polypeptide, means that the polypeptide comprises a Cys residue in a location where there is no Cys in the corresponding wild-type polypeptide. This can be accomplished through routine protein engineering in which a cysteine is substituted for the amino acid that occurs in the wild-type sequence.
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. Unless otherwise stated, “sequence identity” as referred to herein is determined by BLAST (Basic Local Alignment Search Tool), as described in Altschul et al. (1990) J. Mol. Biol. 215:403.
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 asparagine-glutamine.
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.
“T cell” includes all types of immune cells expressing CD3, including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg), and NK-T cells.
The term “immunomodulatory polypeptide” (also referred to herein as a “MOD”), as used herein, means a polypeptide that specifically binds a cognate costimulatory polypeptide on a T cell, thereby providing a signal which, in 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. As discussed herein, a MOD can include, but is not limited to wild-type or variants of wild-type polypeptides such as a cytokine (e.g., IL-2), CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas 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. A MOD of a TMMP can bind a cognate costimulatory polypeptide (i.e., a “co-MOD”) that is present on a target T cell.
As used herein the term “in vivo” refers to any process or procedure occurring inside of the body.
As used herein, “in vitro” refers to any process or procedure occurring outside of the body.
“Heterologous,” as used herein, means a nucleotide or polypeptide that is not found in the native nucleic acid or protein, respectively.
“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 cDNA 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.
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.
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). 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.
The term “binding,” as used herein (e.g., with reference to binding of a TMMP to a polypeptide (e.g., a T-cell receptor) on a T 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 hydrogen-bond interactions, including interactions such as salt bridges and water bridges. “Affinity” refers to the strength of non-covalent binding, increased binding affinity being correlated with a lower KD. “Specific binding” generally refers to binding of a ligand to a moiety that is than its designated binding site or receptor. “Non-specific binding” generally refers to binding of a ligand to a moiety other than its designated binding site or receptor. “Covalent binding” or “covalent bond,” as used herein, refers to the formation of one or more covalent chemical binds between two different molecules.
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 or may not be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or one or more symptoms associated with the disease, e.g., arresting its development; and/or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during and/or after the onset of disease or injury. The treatment of ongoing disease, 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.
The terms “individual,” “subject,” “host,” and “patient,” are used interchangeably 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. Unless otherwise indicated, the terms “individual,” “subject,” “host,” and “patient,” refer to a human.
The terms “antibodies” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAb), single domain antibodies (dAb), single domain heavy chain antibodies, a single domain light chain antibodies, nanobodies, bi-specific antibodies, multi-specific antibodies, and fusion proteins comprising an antigen-binding (also referred to herein as antigen binding) portion of an antibody and a non-antibody protein. The antibodies can be detectably labeled, e.g., with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. The antibodies can be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. Also encompassed by the term are Fab′, Fv, F(ab′)2, and or other antibody fragments that retain specific binding to antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which were produced from a single cell by repetitive cellular replication. That is, the clone of cells only produces a single antibody species. While a monoclonal antibody can be produced using hybridoma production technology, other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). An antibody can be monovalent or bivalent. An antibody can be an Ig monomer, which is a “Y-shaped” molecule that consists of four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.
The term “nanobody” (Nb), as used herein, refers to the smallest antigen binding fragment or single variable domain (VHH) derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. They are derived from heavy chain only antibodies, seen in camelids (Hamers-Casterman et al. (1993) Nature 363:446; Desmyter et al. (1996) Nature Structural Biol. 3:803; and Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1). In the family of “camelids” immunoglobulins devoid of light polypeptide chains are found. “Camelids” comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Llama paccos, Llama glama, Llama guanicoe and Llama vicugna). A single variable domain heavy chain antibody is referred to herein as a nanobody or a VHH antibody.
“Antibody fragments” comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); domain antibodies (dAb; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
“Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRS of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab′ fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subclasses can be further divided into types, e.g., IgG2a and IgG2b.
“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.
As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al (1977) J. Biol. Chem. 252:6609; Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991) (also referred to herein as Kabat 1991); by Chothia et al. (1987) J. Mol. Biol. 196:901 (also referred to herein as Chothia 1987); and MacCallum et al. (1996) J. Mol. Biol. 262:732, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues, which encompass the CDRs, as defined by each of the above cited references are set forth below in Table 1 as a comparison.
1Residue numbering follows the nomenclature of Kabat et al., 1991, supra
2Residue numbering follows the nomenclature of Chothia et al., supra
3Residue numbering follows the nomenclature of MacCallum et al., supra
As used herein, the terms “CDR-L1”, “CDR-L2”, and “CDR-L3” refer, respectively, to the first, second, and third CDRs in a light chain variable region. The terms “CDR-L1”, “CDR-L2”, and “CDR-L3” may be used interchangeably with “VL CDR1,” “VL CDR2,” and “VL CDR3,” respectively. As used herein, the terms “CDR-H1”, “CDR-H2”, and “CDR-H3” refer, respectively, to the first, second, and third CDRs in a heavy chain variable region. The terms “CDR-H1”, “CDR-H2”, and “CDR-H3” may be used interchangeably with “VH CDR1,” “VH CDR2,” and “VH CDR3,” respectively. As used herein, the terms “CDR-1”, “CDR-2”, and “CDR-3” refer, respectively, to the first, second and third CDRs of either chain's variable region.
As used herein, the term “framework,” when used in reference to an antibody variable region, is intended to mean all amino acid residues outside the CDR regions within the variable region of an antibody. A variable region framework is generally a discontinuous amino acid sequence between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs. As used herein, the term “framework region” is intended to mean each domain of the framework that is separated by the CDRs.
Unless indicated otherwise, the term “substantially” is intended to encompass both “wholly” and “largely but not wholly”. For example, an Ig Fc that “substantially does not induce ADCC” means an Ig Fc that induces no ADCC at all or that largely does not induce ADCC.
As used herein, the term “about” used in connection with an amount indicates that the amount can vary by 10% of the stated amount. For example, “about 100” means an amount of from 90-110. Where about is used in the context of a range, the “about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and “about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.
As used herein, the term “MHC heavy chain polypeptide” means collectively the domains of an MHC heavy chain polypeptide that are present in a TMMP. For example, an MHC heavy chain polypeptide can comprise α1, α2 and α3 domains.
Before the present disclosure is further described, it is to be understood that this disclosure 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 will be limited only by the appended claims.
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 disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, 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 disclosure.
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 disclosure 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 disclosure, 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.
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 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 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.
It is appreciated that certain features of the disclosure, 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 disclosure, 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 disclosure are specifically embraced by the present disclosure 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 disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
The publications discussed herein are provided solely for their disclosure. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
The present disclosure provides T-cell modulatory multimeric polypeptides (TMMPs) that comprise (i) an optional immunomodulatory polypeptide such as a variant IL-2 polypeptide, (ii) class I HLA major histocompatibility complex (MHC) polypeptides (a class I HLA heavy chain polypeptide and a f2 microglobulin polypeptide), (iii) a peptide that presents an epitope to a T-cell receptor, which together with the class I MHC polypeptides forms a peptide-MHC complex (pMHC), (iv) a tumor-targeting polypeptide, and (v) an optional Ig Fc polypeptide or other scaffold.
A TMMP is useful for modulating the activity of a T cell, and for modulating an immune response in an individual. Importantly, the TMMPs disclosed herein are useful for “redirecting” a patient's repertoire of anti-pathogenic (e.g., antiviral) T cells to attack and kill cancer cells. The TMMPs achieve this result by (i) binding to the patient's cancer cells via the TMMP's tumor-targeting polypeptide (TTP) that is specific for and binds an antigen on the cancer cell, and (ii) presenting a pMHC that mimics a pMHC on the surface of a pathogen-infected cell, e.g., a cell infected by a virus, bacteria or other microorganism that can cause disease. In this way, TMMPs effectively “paint” the cancer cells to make them appear like pathogen-infected cells. The “painted” cancer cells are then susceptible to being attacked and killed by the patient's existing repertoire of anti-pathogenic T cells that have a T cell receptor (TCR) that recognizes and binds to the pMHC presented by the TMMP.
For example, TMMPs can have a TTP that binds to an antigen on a cancer cell, and a pMHC that mimics the pMHC of a cell that has been infected with a virus such as a SARS-CoV-2 virus. The patient's existing repertoire of T cells against a SARS-CoV-2 virus-infected cells are thus re-directed to the patient's cancer cells where the T cells can be activated through binding of the TCR of T cells to the pMHC of the TMMP. Activation of the T cells can lead to release of their cytotoxic components and killing of the cancer cells. In such cases, the patient's repertoire of T cells specific the SARS-CoV-2 virus can be the result of one or more of a prior SARS-CoV-2 infection, vaccination with a vaccine that elicits T cells having a TCR that is specific for the pMHC of the TMMP, and/or prior treatment with a T cell modulatory protein designed to prime and/or increase the patient's T cells that have a TCR that specifically binds the pMHC of the TMMP.
The TMMPs disclosed herein are useful for any patient having a hematological cancer or solid tumor, including patients whose cancers have evaded their immune system through HLA loss, which prevents the patient's own cancer-specific T cells from recognizing and killing the cancer cells.
The present disclosure provides a T-cell modulatory multimeric polypeptide (TMMP) comprising: a) a first polypeptide; and b) a second polypeptide, wherein the TMMP comprises: a peptide epitope (defined below); a first major histocompatibility complex (MHC) polypeptide; a second MHC polypeptide; one or more immunomodulatory polypeptides; an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold; and a tumor targeting polypeptide (TTP).
In some cases, a TMMP comprises a heterodimer (comprises two different separate polypeptide chains) comprising: a) a first polypeptide; and b) a second polypeptide, wherein the TMMP comprises: a peptide epitope; a first MHC polypeptide; a second MHC polypeptide; one or more immunomodulatory polypeptides; an Ig Fc polypeptide or a non-Ig scaffold; and a TTP. A TMMP can comprise a single heterodimer. A TMMP can comprise two copies of a heterodimer; i.e., a TMMP can be a homodimer of a heterodimer. The two copies of a heterodimer can be linked to one another, e.g., by disulfide bonds (e.g., disulfide bonds between Ig Fc polypeptides present in the heterodimers)
In some cases, a TMMP comprises one or more heterodimers, each 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 a peptide epitope (defined below), wherein the first polypeptide and/or the second polypeptide comprises one or more immunomodulatory polypeptides that can be the same or different; and an Ig Fc polypeptide or a non-Ig scaffold. The first or the second polypeptide also includes a TTP. In some cases, the TTP is at the C-terminus of the Ig Fc polypeptide or the non-Ig scaffold. In some cases, the TTP is at the N-terminus of the polypeptide that comprises the Ig Fc polypeptide. In some cases, the TTP is at the C-terminus of the polypeptide that comprises the peptide epitope.
The peptide epitope present in a TMMP presents a SARS-CoV-2 peptide (e.g., a SARS-CoV-2 encoded peptide). As used herein, the term “peptide epitope” means a peptide that, when complexed with MHC polypeptides, presents an epitope to a T-cell receptor (TCR). When complexed with MHC polypeptides, a peptide epitope can present one or more epitopes to one or more TCRs.
In some cases, a TMMP includes: i) a SARS-CoV-2 peptide; and iii) a TTP that targets a cancer-associated antigen. Such a TMMP binds a cancer cell that expresses the cancer-associated antigen targeted by the TTP. The TMMP modulates the activity of a T-cell specific for the virus epitope present in the TMMP. For example, in some cases, the TMMP increases proliferation and/or cytotoxic activity of a T-cell specific for the virus epitope present in the TMMP. Contacting a T-cell specific for the virus epitope present in the TMMP can increase cytotoxic activity of the T cell toward a cancer cell expressing the cancer-associated antigen that is targeted by the TTP present in the TMMP.
TMMPs comprising a pMHC that has a SARS-CoV-2 epitope are particularly useful because the high rate of SARS-CoV-2 infections worldwide and the availability of commercial vaccines means that most patients either already have, or can be induced to have through vaccination, a repertoire of T cells that can recognize a pMHC having a SARS-CoV-2 epitope. As noted above, however, a pMHC of a TMMP alternatively could present a peptide epitope from other virus or pathogenic microorganisms against which the patient has an existing repertoire of T cells. For example, the pMHC could present an epitope that would be recognized by T cells that result from administration of commonly available vaccines that provide immunity against pathogens such as tetanus, flu or other available vaccines. There are numerous commercially available vaccines against pathogens, e.g., Cholera, Dengue, Diphtheria, Hepatitis, Haemophilus influenzae type b (Hib), Human papillomavirus (HPV), Influenza, Japanese encephalitis, Malaria, Measles, Meningococcal meningitis, Mumps, Pertussis, Pneumococcal disease, Poliomyelitis, Rabies, Rotavirus, Rubella, Tetanus, Tick-borne encephalitis, Tuberculosis, Typhoid, Varicella and Yellow Fever, with numerous other in development, e.g., Enterotoxigenic Escherichia coli, Group B Streptococcus (GBS), Herpes Simplex Virus, HIV-1, Malaria, Neisseria gonorrhoeae, Nontyphoidal Salmonella Disease, Norovirus, Paratyphoid fever, Respiratory Syncytial Virus (RSV), Schistosomiasis Disease, Shigella, Group A Streptococcus (GAS), Tuberculosis, and other Influenza vaccines. Alternatively, (or additionally), prior to administering the TMMP, one or more doses of a T cell modulatory polypeptide can be administered to the patient, in order to prime and activate and/or induce proliferation of (and thereby increase the number of) CD8+ T cells that recognize the desired pMHC in the TMMP comprising the TTP. See, e.g., the T cell modulatory polypeptides described in WO2021/195108, published Sep. 30, 2021 (Cue Biopharma, Inc.), WO 2021/195411, published Sep. 30, 2021 (Cue Biopharma, Inc.), and WO 2022/015880, published Jan. 20, 2022 (Cue Biopharma, Inc.).
In some cases, a TMMP comprises one or more heterodimers, each heterodimer comprising: a) a first polypeptide comprising: i) a peptide epitope (a SARS-CoV-2 peptide); and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; c) one or more immunomodulatory polypeptides, where the first and/or the second polypeptide comprises the at least one (i.e., one or more) immunomodulatory polypeptide; d) an Ig Fc polypeptide or a non-Ig scaffold, where the first and/or the second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and e) a polypeptide that targets a cancer cell (a tumor-targeting polypeptide; “TTP”). In some cases, at least one of the one or more immunomodulatory polypeptides comprises an amino acid sequence of a wild-type immunomodulatory polypeptide. In some cases, at least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that binds to but 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. In some cases, the peptide epitope present in a TMMP presents an infectious disease-associated epitope (e.g., a virus-encoded peptide).
A TMMP comprises a tumor targeting polypeptide, i.e., a polypeptide that targets a cancer-associated epitope displayed on the surface of a cancer cell.
In some cases, a TMMP is:
In some cases, an immunomodulatory polypeptide present in a TMMP comprises a wild-type (naturally-occurring) amino acid sequence.
In some cases, an immunomodulatory polypeptide present in a TMMP binds to its cognate co-immunomodulatory polypeptide with an affinity that is less than the affinity of a corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide.
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. For example, a TMMP binds selectively to a first T cell that displays both: i) a TCR specific for the epitope present in the TMMP; and ii) a co-immunomodulatory 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 co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP.
In some cases, a TMMP, 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, 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 both: i) a TCR specific for the peptide epitope present in the TMMP; ii) a co-immunomodulatory 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 a T cell can include one or more of: i) activating a cytotoxic (e.g., CD8+) T cell; ii) inducing cytotoxic activity of a cytotoxic (e.g., CD8+) T cell; iii) inducing production and release of a cytotoxin (e.g., a perforin; a granzyme; a granulysin) by a cytotoxic (e.g., CD8+) T cell; iv) inhibiting activity of an autoreactive T cell; and the like. As used herein, selectively modulating refers to inducing an epitope-specific T cell response that is greater than the epitope-non-specific T cell response.
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. Thus, for example, a TMMP 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 co-immunomodulatory 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.
Binding affinity between an immunomodulatory polypeptide and its cognate co-immunomodulatory polypeptide can be determined by bio-layer interferometry (BLI) as described in published PCT Application WO 2020/243315 (Cue Biopharma, Inc.).
A TMMP can be dimerized; i.e., the present disclosure provides a multimeric polypeptide comprising a dimer of a TMMP. 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 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 β2-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 co-immunomodulatory 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), TGFβ, CD70, and ICAM. Examples, of suitable MHC polypeptides, immunomodulatory polypeptides, and peptide epitopes are described below. The first and/or the second polypeptide comprises: i) an Ig Fc polypeptide or a non-Ig scaffold; and ii) a tumor-targeting polypeptide.
In some cases, the first polypeptide and the second polypeptide of a TMMP are linked to one another by at least two disulfide bonds (i.e., two interchain disulfide bonds). Examples of such multiple disulfide-linked TMMP are depicted schematically in
As noted above, in some cases, the first polypeptide and the second polypeptide of a TMMMP are linked to one another by at least two disulfide bonds (i.e., two interchain disulfide bonds). For example, in some instances, the first polypeptide and the second polypeptide of a TMMP are linked to one another by 2 interchain disulfide bonds. As another example, in some instances, the first polypeptide and the second polypeptide of a TMMP are linked to one another by 3 interchain disulfide bonds. As another example, in some instances, the first polypeptide and the second polypeptide of a TMMP are linked to one another by 4 interchain disulfide bonds.
In some cases where a peptide epitope in a first polypeptide of a TMMP is linked to a 32M polypeptide by a linker comprising a Cys, at least one of the at least two disulfide bonds links a Cys in the linker to a Cys in an MHC Class I heavy chain in the second polypeptide. In some cases, where a peptide epitope in a first polypeptide of a TMMP is linked to an MHC Class I heavy chain polypeptide by a linker, at least one of the at least two disulfide bonds links a Cys in the linker to a Cys in a β2M polypeptide present in the second polypeptide.
In some cases, a T-cell modulatory multimeric polypeptide comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; c) at least one immunomodulatory polypeptide, where the first and/or the second polypeptide comprises the at least one immunomodulatory polypeptide; d) an Ig Fc polypeptide or a non-Ig scaffold, where the first and/or the second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and e) a TTP, where the first and/or the second polypeptide comprises the TTP; and where the heterodimer comprises at least two disulfide bonds (e.g., two disulfide bonds) between the first polypeptide and the second polypeptide (e.g., the heterodimer comprises: i) a first disulfide bond linking the first polypeptide and the second polypeptide; and ii) a second disulfide bond linking the first polypeptide and the second polypeptide). Expressed another way, the first polypeptide comprises a first Cys residue that forms a disulfide bond (a first disulfide bond) with a first Cys residue in the second polypeptide; and the first polypeptide comprises a second Cys residue that forms a disulfide bond (a second disulfide bond) with a second Cys residue in the second polypeptide.
In some cases, a TMMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide epitope; ii) a peptide linker; and iii) a β2M polypeptide; and b) a second polypeptide comprising an MIHC Class I heavy chain polypeptide, where one or both of the first and the second polypeptides comprises at least one immunomodulatory polypeptide, where the TMMP comprises: a) a first disulfide linkage between: i) a Cys present in the linker between the peptide epitope and the β2M polypeptide; and ii) a first Cys introduced into the MHC Class I heavy chain polypeptide; and b) at least a second disulfide linkage between the first polypeptide and the second polypeptide, where the at least a second disulfide linkage is between: i) a Cys in the first polypeptide that is C-terminal to the Cys present in the linker; and ii) a Cys in the second polypeptide that is C-terminal to the first Cys introduced into the MHC Class I heavy chain polypeptide. As noted above, the TMMP also includes: i) an Ig Fe polypeptide or a non-Ig scaffold; and ii) a TTP.
A multiple disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) can comprise, for example: a) a first polypeptide comprising: i) a peptide epitope; and ii) a first MHC polypeptide, where the first polypeptide comprises a peptide linker between the peptide and the first MHC polypeptide, where the peptide linker comprises a Cys residue, and where the first MHC polypeptide is a β2M polypeptide that comprises an amino acid substitution that introduces a Cys residue; b) and a second polypeptide comprising a second MHC polypeptide, where the second MHC polypeptide is a Class I heavy chain comprising a Y84C substitution and an A236C substitution, based on the amino acid numbering of HLA-A*0201 (depicted in
As discussed above, a TMMP comprises a Betacoronavirus (e.g., SARS-CoV-2) peptide that is typically at least about 4 amino acids in length, and presents a SARS-CoV-2 epitope to a T cell when in an MHC/peptide complex (e.g., an HLA/peptide complex).
A SARS-CoV-2 peptide present in a TMMP can have a length of at least 4 amino acids, e.g., from 4 amino acids to about 25 amino acids in length (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa, including within a range of from 4 to 20 amino acids, from 6 to 18 amino acids, from 8 to 15 amino acids, from 8 to 12 amino acids, from 9-11 amino acids, from 9-10 amino acids, from 5 to 10 amino acids, from 10-15 amino acids, from 10 to 20 amino acids, and from 15 to 25 amino acids in length), for example, lengths of 9, 10, 11, 12, 13 or 14 amino acids.
A SARS-CoV-2 epitope present in a TMMP is a peptide specifically bound by a T-cell, i.e., the epitope is specifically bound by an epitope-specific T cell. An epitope-specific T cell binds an epitope having a reference amino acid sequence, but does not substantially bind an epitope that differs from the reference amino acid sequence. For example, an epitope-specific T cell binds an epitope having a reference amino acid sequence, and binds an epitope that differs from the reference amino acid sequence, if at all, with an affinity that is less than 10−6 M, less than 10−5 M, or less than 10−4 M. An epitope-specific T cell can bind an epitope for which it is specific with an affinity of at least 10−7 M, at least 10−8 M, at least 10−9 M, or at least 10−10.
The peptide epitope present in a TMMP is a peptide of a Betacoronavirus-encoded polypeptide. In some cases, the peptide epitope is a SARS-CoV-2 peptide (i.e., a peptide of a SARS-CoV-2-encoded polypeptide). In some cases, the peptide epitope is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded surface glycoprotein. In some cases, the peptide epitope is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded membrane glycoprotein. In some cases, the peptide epitope is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded nucleocapsid phosphoprotein.
In some cases, a peptide present in a TMMP is a peptide of from 4 amino acids (aa) to 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, 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 any one of the SARS-CoV-2 polypeptides depicted in
In some cases, a peptide present in a TMMP is any one of the peptides depicted in
In some cases, the peptide epitope is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded surface glycoprotein. In some cases, a peptide present in a TMMP is a peptide of from 4 amino acids (aa) to 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, 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 SARS-CoV-2 surface glycoprotein depicted in
In some cases, the peptide epitope is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded surface glycoprotein, where the SARS-CoV-2 is the omicron variant (also known as the B1.1.529 variant). In some cases, a peptide present in a TMMP is a peptide of from 4 amino acids (aa) to 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, 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 SARS-CoV-2 surface glycoprotein depicted in
In some cases, a peptide present in a TMMP is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded surface glycoprotein and is selected from the group consisting of: NLTTRTQL (SEQ ID NO:533), LPPAYTNSF (SEQ ID NO:590), KVFRSSVLH (SEQ ID NO:288), LPFFSNVTW (SEQ ID NO:591), PFFSNVTWF (SEQ ID NO:388), RFDNPVLPF (SEQ ID NO:389), LPFNDGVYF (SEQ ID NO:475), GVYFASTEK (SEQ ID NO:289), TEKSNIIRGW (SEQ ID NO:711), TLDSKTQSL (SEQ ID NO:534), GVYYHKNNK (SEQ ID NO:290), YYHKNNKSW (SEQ ID NO:412), VYSSANNCTF (SEQ ID NO:390), FEYVSQPFL (SEQ ID NO:661), EYVSQPFLM (SEQ ID NO:391), FVFKNIDGY (SEQ ID NO:592), TPINLVRDL (SEQ ID NO:476), LPQGFSAL (SEQ ID NO:477), LPIGINITRF (SEQ ID NO:593), INITRFQTL (SEQ ID NO:535), LLALHRSYL (SEQ ID NO:536), WTAGAAAYY (SEQ ID NO:145), YYVGYLQPRTF (SEQ ID NO:392), YLQPRTFLL (SEQ ID NO:218), YLQPRTFL (SEQ ID NO:537), SETKCTLKSF (SEQ ID NO:712), TLKSFTVEK (SEQ ID NO:291), QPTESIVRF (SEQ ID NO:594), RFPNITNLCPF (SEQ ID NO:413), GEVFNATRF (SEQ ID NO:662), NATRFASVY (SEQ ID NO:595), LYNSASFSTF (SEQ ID NO:393), NSASFSTFK (SEQ ID NO:329), RQIAPGQTGK (SEQ ID NO:292), KIADYNYKL (SEQ ID NO:219), NYNYLYRLF (SEQ ID NO:394), RLFRKSNLK (SEQ ID NO:293), KPFERDISTEI (SEQ ID NO:478), YFPLQSYGF (SEQ ID NO:395), QPYRVVVL (SEQ ID NO:479), PYRVVVLSF (SEQ ID NO:396), GPKKSTNLV (SEQ ID NO:480), TSNQVAVLY (SEQ ID NO:146), VYSTGSNVF (SEQ ID NO:397), AEHVNNSY (SEQ ID NO:721), IPIGAGICASY (SEQ ID NO:596), SPRRARSVA (SEQ ID NO:481), VASQSIIAY (SEQ ID NO:597), SIIAYTMSL (SEQ ID NO:220), LGAENSVAY (SEQ ID NO:598), AYSNNSIAIPTNF (SEQ ID NO:414), IPTNFTISV (SEQ ID NO:482), TEILPVSMTK (SEQ ID NO:330), QEVFAQVKQIY (SEQ ID NO:713), KQIYKTPPIK (SEQ ID NO:294), IYKTPPIKDF (SEQ ID NO:398), LLFNKVTLA (SEQ ID NO:221), TLADAGFIK (SEQ ID NO:295), LADAGFIKQY (SEQ ID NO:147), ADAGFIKQY (SEQ ID NO:714), VLPPLLTDEMIAQY (SEQ ID NO:148), IPFAMQMAY (SEQ ID NO:599), SSTASALGK (SEQ ID NO:331), VLNDILSRL (SEQ ID NO:222), RLDKVEAEV (SEQ ID NO:223), VEAEVQIDRL (SEQ ID NO:663), AEVQIDRLI (SEQ ID NO:664), LITGRLQSL (SEQ ID NO:538), RLQSLQTYV (SEQ ID NO:224), AEIRASANL (SEQ ID NO:665), ASANLAATK (SEQ ID NO:296), HLMSFPQSA (SEQ ID NO:225), FPQSAPHGVVF (SEQ ID NO:600), APHGVVFL (SEQ ID NO:483), VTYVPAQEK (SEQ ID NO:297), TYVPAQEKNF (SEQ ID NO:399), REGVFVSNGTHW (SEQ ID NO:715), GTHWFVTQR (SEQ ID NO:332), TVYDPLQPELDSFK (SEQ ID NO:333), KEIDRLNEV (SEQ ID NO:666), QELGKYEQYIKW (SEQ ID NO:716), YEQYIKWPW (SEQ ID NO:717), QYIKWPWYI (SEQ ID NO:400), FIAGLIAIV (SEQ ID NO:226), and SEPVLKGVKL (SEQ ID NO:484).
In some cases, the peptide epitope is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded membrane glycoprotein. In some cases, a peptide present in a TMMP is a peptide of from 4 amino acids (aa) to 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, 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 SARS-CoV-2 membrane glycoprotein depicted in
In some cases, a peptide present in a TMMP is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded membrane glycoprotein and is selected from the group consisting of: GTITVEELK (SEQ ID NO:302), EELKKLLEQW (SEQ ID NO:671), KLLEQWNLV (SEQ ID NO:155), FAYANRNRF (SEQ ID NO:544), YANRNRFLY (SEQ ID NO:545), SYFIASFRLF (SEQ ID NO:338), RLFARTRSM (SEQ ID NO:491), VPLHGTIL (SEQ ID NO:427), SELVIGAVIL (SEQ ID NO:602), HLRIAGHHL (SEQ ID NO:492), RIAGHHLGR (SEQ ID NO:233), KEITVATSRTL (SEQ ID NO:603), ATSRTLSYYK (SEQ ID NO:303), ASQRVAGDSGFAAY (SEQ ID NO:101), and VAGDSGFAAY (SEQ ID NO:102).
In some cases, the peptide epitope is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded nucleocapsid phosphoprotein. In some cases, a peptide present in a TMMP is a peptide of from 4 amino acids (aa) to 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, 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 SARS-CoV-2 nucleocapsid phosphoprotein depicted in
In some cases, the peptide epitope is a SARS-CoV-2 peptide from a SARS-CoV-2-encoded nucleocapsid phosphoprotein and is selected from the group consisting of: LPNNTASWF (SEQ ID NO:567), KFPRGQGVPI (SEQ ID NO:454), NTNSSPDDQIGYY (SEQ ID NO:119), SPRWYFYYL (SEQ ID NO:455), LLLDRLNQL (SEQ ID NO:192), KAYNVTQAF (SEQ ID NO:568), QELIRQGTDYKHW (SEQ ID NO:690), ASAFFGMSR (SEQ ID NO:315), SRIGMEVTPSGTW (SEQ ID NO:691), GMEVTPSGTWL (SEQ ID NO:692), TPSGTWLTY (SEQ ID NO:569), AYKTFPPTEPK (SEQ ID NO:316), and LPAADLDDF (SEQ ID NO:570).
In some cases, the peptide epitope is a SARS-CoV-2 peptide depicted in
In some cases, the peptide epitope is RLQSLQTYV (SEQ ID NO:224). In some cases, the peptide epitope is YLQPRTFLL (SEQ ID NO:218).
In some cases, the peptide epitope is one that can be presented in a complex with a β2M polypeptide and an HLA-E polypeptide. As one non-limiting example, in some cases, the peptide epitope is a peptide of a SARS-CoV-2 Nsp13 (helicase) polypeptide (see, e.g.,
As noted above, a TMMP 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., β-2 microglobulin and MHC class I heavy chain).
In some cases, the first MHC polypeptide is an MHC class I beta-2 microglobulin (β2M) 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 β2M polypeptide. In some cases, both the β2M 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 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 heavy chain present in a TMMP 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 includes only the a1, a2, and a3 domains of an MHC class I heavy chain polypeptide. In some cases, the MHC class I heavy chain present in a TMMP 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 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 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 any of the human HLA heavy chain polypeptides depicted in
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 β2-microglobulin polypeptide, or a Class I HLA heavy chain polypeptide. Class I HLA heavy chain polypeptides include HLA-A heavy chain polypeptides, HLA-B heavy chain polypeptides, HLA-C heavy chain polypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chain polypeptides, and HLA-G heavy chain polypeptides.
In some cases, a TMMP 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 include, but are not limited to, the alleles: A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and A*3401. Any of those alleles may comprise a mutation at one or more of positions 84, 139, and 236 (as shown in
In some cases, an MHC class I heavy chain polypeptide of a TMMP 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 amino acid sequence depicted in
To facilitate the formation of such disulfide bonds, one or more non-naturally occurring Cys residues can be provided in the heavy chain polypeptide. For example, the MHC class I heavy chain 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) the HLA-A02 (Y84C; A236C) amino acid sequence depicted in
As noted above, in some cases the MHC class I heavy chain 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 the HLA-A02 (Y84A; A236 wild-type) amino acid sequence depicted in
In some cases, an MHC class I heavy chain polypeptide of a TMMP 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 amino acid sequence depicted in
In some cases, an MHC class I heavy chain polypeptide of a TMMP 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 amino acid sequence depicted in
In some cases, an MHC class I heavy chain polypeptide of a TMMP 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 amino acid sequence depicted in
With regard to
With regard to
With regard to
In some cases, a TMMP comprises a non-classical MHC class I heavy chain polypeptide. Among the non-classical HLA heavy chain polypeptides, or portions thereof, that may be that may be incorporated into a TMMP include, but are not limited to, those of HLA-E, -F, and -G alleles. Amino acid sequences for HLA-E, -F, and -G heavy chain polypeptides, (and the HLA-A, B and C alleles) may be found on the world wide web hla.alleles.org/nomenclature/index.html, the European Bioinformatics Institute (www(dot)ebi(dot)ac(dot)uk), which is part of the European Molecular Biology Laboratory(EMBL), and at the National Center for Biotechnology Information (www(dot)ncbi(dot)nlm(dot)nih(dot)gov).
Non-limiting examples of suitable HLA-E alleles include, but are not limited to, HLA-E*0101 (HLA-E*01:01:01:01), HLA-E*01:03(HLA-E*01:03:01:01), HLA-E*01:04, HLA-E*01:05, HLA-E*01:06, HLA-E*01:07, HLA-E*01:09, and HLA-E*01:10. Of these, isoforms HLA-E*0101 and HLA-E*01.03 are of particular note since these are highly prevalent alleles, and differ by only 1 amino acid (Arg or Gly at position 107). For example, amino acid sequences of suitable HLA-E heavy chain polypeptides are provided in
Non-limiting examples of suitable HLA-F alleles include, but are not limited to, HLA-F*0101 (HLA-F*01:01:01:01), HLA-F*01:02, HLA-F*01:03(HLA-F*01:03:01:01), HLA-F*01:04, HLA-F*01:05, and HLA-F*01:06. Non-limiting examples of suitable HLA-G alleles include, but are not limited to, HLA-G*0101 (HLA-G*01:01:01:01), HLA-G*01:02, HLA-G*01:03(HLA-G*01:03:01:01), HLA-G*01:04 (HLA-G*01:04:01:01), HLA-G*01:06, HLA-G*01:07, HLA-G*01:08, HLA-G*01:09: HLA-G*01:10, HLA-G*01:10, HLA-G*01:11, HLA-G*01:12, HLA-G*01:14, HLA-G*01:15, HLA-G*01:16, HLA-G*01:17, HLA-G*01:18: HLA-G*01:19, HLA-G*01:20, and HLA-G*01:22. Of these, isoforms HLA-G*0101 (HLA-G*01:01:01:01) and HLA-G*01:04 (HLA-G*01:04:01:01) are of particular note since these are highly prevalent alleles. For example, amino acid sequences of suitable HLA-G heavy chain polypeptides are provided in
Consensus sequences for those HLA E, -F and -G alleles without all, or substantially all, of the leader, transmembrane and cytoplasmic sequences are provided in
Any of the above-mentioned HLA-E, -F, and/or -G alleles may comprise a substitution at one or more of positions 84, 139 and/or 236 as shown in
A β2-microglobulin (β2M) polypeptide of a TMMP can be a human β2M polypeptide, a non-human primate β2M polypeptide, a murine β2M polypeptide, and the like. In some instances, a β2M 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 the amino acid sequence depicted in
In some cases, an MHC polypeptide present in a TMMP 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 can form a disulfide bond with a naturally occurring or non-naturally occurring cysteine residue present in the MHC heavy chain of the TMMP. As used herein, a reference to a “non-naturally occurring Cys residue” in an MHC class I polypeptide means that the polypeptide comprises a Cys residue in a location where there is no Cys in the corresponding wild-type polypeptide. This can be accomplished through routine protein engineering in which a cysteine is substituted for the amino acid that occurs in the wild-type sequence.
In some cases, a β2M polypeptide present in a TMMP 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 the amino acid sequence depicted in
In some cases, an immunomodulatory polypeptide (“MOD”) present in a TMMP is a wild-type (“wt”) MOD. As discussed above, in other cases, a MOD present in a TMMP is a variant of a wt. MOD that has reduced affinity for a co-MOD compared to the affinity of a corresponding wild-type MOD for the co-MOD. Suitable MODs that exhibit reduced affinity for a co-MOD can have from 1 amino acid (aa) to 20 aa differences from a wild-type MOD.
As discussed above, a MOD may comprise a variant of a wt immunomodulatory polypeptide that may exhibit reduced binding to its co-MOD, including e.g., reduced binding to one or more chains or domains of the co-MOD. For example, a variant MOD present in a TMMP may bind its co-MOD 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 MOD for the co-MOD.
Exemplary pairs of MODs and their co-MODs include, but are not limited to those set out in Table 2, below:
One or more MODs can be present in a TMMP at any of a variety of positions. For example, in some cases, a MOD (or multiple MODs) can be: i)C-terminal to the MHC class I heavy chain and N-terminal to the Ig Fc polypeptide; in other words, between the MHC class I heavy chain polypeptide and the Ig Fc polypeptide; 2)C-terminal to the Ig Fc polypeptide; or 3)N-terminal to the peptide epitope.
Immunomodulatory polypeptides and variants, including reduced affinity variants, such as PD-L1, CD80, CD86, 4-1BB1L and IL-2 are described in the published literature, e.g., published PCT application WO2020132138A1 and WO2019/051091, the disclosures of which as they pertain to MODs and specific variant MODs of PD-L1, CD80, CD86, 4-1BB1L, IL-2 are expressly incorporated herein by reference, including specifically paragraphs [00260]-[00455] of WO2020132138A1 and paragraphs [00157]-[00352] of WO2019/051091.
Of specific interest are MODs that are variants of the cytokine IL-2. Wild-type IL-2 binds to IL-2 receptor (IL-2R) on the surface of a T cell. Wild-type IL-2 has a strong affinity for IL-2R and will bind to activate most or substantially all CD8+ T cells. For this reason, synthetic forms of wild type IL-2 such as the drug Aldesleukin (trade name Proleukin®) are known to have severe side-effects when administered to humans for the treatment of cancer because the IL-2 indiscriminately activates both target and non-target T cells.
An IL-2 receptor is in some cases a heterotrimeric polypeptide comprising an alpha chain (IL-2Ra; also referred to as CD25), a beta chain (IL-2Rβ; also referred to as CD122; and a gamma chain (IL-2Ry; also referred to as CD132). Amino acid sequences of human IL-2, human IL-2Ra, IL2RP, and IL-2Rγ are known. See, e.g., published PCT applications WO2020132138A1 and WO2019/051091, discussed above. For example, a wild-type IL-2 polypeptide can have the amino acid sequence depicted in
In some cases, an IL-2 variant MOD of this disclosure exhibits decreased binding to IL-2Ra, thereby minimizing or substantially reducing the activation of Tregs by the IL-2 variant. Alternatively, or additionally, in some cases, an IL-2 variant MOD of this disclosure exhibits decreased binding to IL-2RP such that the IL-2 variant MOD exhibits an overall reduced affinity for IL-2R. In some cases, an IL-2 variant MOD of this disclosure exhibits both properties, i.e., it exhibits decreased or substantially no binding to IL-2Ra, and also exhibits decreased binding to IL-2RP such that the IL-2 variant polypeptide exhibits an overall reduced affinity for IL-2R. For example, IL-2 variants having substitutions at H16 and F42 have shown decreased binding to IL-2Ra and IL-2RP. See, Quayle et al., Clin Cancer Res; 26(8) Apr. 15, 2020, which discloses that the binding affinity of an IL-2 polypeptide with H16A and F42A substitutions for human IL-2Ra and IL-2RP was decreased 110- and 3-fold, respectively, compared with wild-type IL2 binding, predominantly due to a faster off-rate for each of these interactions. TMMPs comprising such variants, including variants that exhibit decreased binding to IL-2Ra and IL-2Rβ, have shown the ability to preferentially bind to and activate IL-2 receptors on T cells that contain the target TCR that is specific for the peptide epitope on the TMMP, and are thus less likely to deliver IL-2 to non-target T cells, i.e., T cells that do not contain a TCR that specifically binds the peptide epitope on the TMMP. That is, the binding of the IL-2 variant MOD to its costimulatory polypeptide on the T cell is substantially driven by the binding of the MHC-epitope moiety rather than by the binding of the IL-2. In some cases, an IL-2 variant MOD of this disclosure exhibits decreased binding to IL-2Rγ. This decreased binding to IL-2Ry may be in addition to the decreased binding to IL-2Rα and/or IL-2Rβ.
Suitable IL-2 variant MODs thus include a polypeptide that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the wild-type IL-2 amino acid sequence depicted in
Mutations that can reduce binding of IL-2 to IL-2Ra include substitutions at one or more of amino acids R38, F42, K43, Y45, E62, P65, E68, V69, and L72. For example, an IL-2 variant can comprise substitutions at one, two, three, four, five or more of the foregoing amino acids. Exemplary substitutions include the following from Table 3:
As noted above, a substitution of F42 with an amino acid other than Phe, e.g., Ala, has been shown to substantially reduce the binding of an IL-2 variant to IL-2Ra. See Quayle et al., Clin Cancer Res 2020; 26:1953-64, which reported that an F42A substitution caused a 110-fold decrease in the binding to IL-2Ra. The binding to IL-2Ra is thus substantially abrogated by this mutation.
Mutations that can reduce binding of IL-2 to IL-2Rβ include substitutions at one or more of amino acids E15, H16, L19, D20, D84, S87, N88, V91, I92. For example, an IL-2 variant can comprise substitutions at one, two, three, four, five or more of the foregoing amino acids. Exemplary substitutions include the following from Table 4:
As noted above, a substitution of H16 with an amino acid other than His, e.g., Ala, has been shown to reduce the binding of an IL-2 variant to IL-2RP3. See Quayle et al., Clin Cancer Res 2020; 26:1953-64, which reported that an H16A substitution caused a 3-fold decrease in the binding to IL-2Rβ.
Some exemplary combinations of mutations that reduce binding of an IL-2 variant polypeptide to IL-2Ra and IL-2Rβ include the following from Table 5:
Exemplary IL-2 variant polypeptide amino acid sequences comprise an amino acid sequence having at least about least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to any one of the following IL-2 variant amino acid sequences (I)-(XVIII):
In any of the foregoing IL-2 variant amino acid sequences (I)-(XVIII), the amino acid sequence may comprise one or more additional mutations that reduce the binding of the IL-2 variant to IL-2Rα and/or IL-2Rβ (as compared to the binding affinity of wild-type IL-2 to IL-2Rα and/or IL-2RP).
The determination of binding affinity may be carried out by known methods.
In some cases, a suitable variant IL-2 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: APTSSSTKKT QLQLEX1LLLD LQMILNGINN YKNPKLTRML TX2KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:1243), i.e., the variant IL-2 polypeptide has the amino acid sequence of wild-type IL-2 but with H16 and F42 substitutions (X1 and X2, shown in bold). As noted above, the histidine at position 16 can be substituted with, e.g., Ala, Glu, Thr, or Asp, but any of the following substitutions also may be employed: Gly, Val, Leu, Ile, Arg, Asn, Cys, Glu, Gln, Ile, Lys, Met, Phe, Pro, Ser, Tyr, or Trp. The phenylalanine at position 42 can be substituted with, e.g., Ala or Lys, but any of the following substitutions also may be employed: Met, Pro, Ser, Thr, Trp, Tyr, or Val.
In some cases, a variant IL-2 polypeptide present in a TMMP comprises the amino acid sequence: APTSSSTKKT QLQLEALLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:878).
In some cases, a variant IL-2 polypeptide present in a TMMP comprises the amino acid sequence: APTSSSTKKT QLQLETLLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:925). In some cases, a TMMP comprises two copies of such a variant IL-2 polypeptide.
In some cases, a variant IL-2 polypeptide present in a TMMP comprises the amino acid sequence: APTSSSTKKT QLQLEELLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:1244). In some cases, a TMMP comprises two copies of such a variant IL-2 polypeptide.
In some cases, a variant IL-2 polypeptide present in a TMMP comprises the amino acid sequence: APTSSSTKKT QLQLEDLLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:1245). In some cases, a TMMP comprises two copies of such a variant IL-2 polypeptide.
As further examples of MODS, in some cases, a MOD present in a TMMP is a 4-1BBL polypeptide. In some cases, a 4-1BBL polypeptide of a TMMP 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 the following 4-1BBL amino acid sequence: DPAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ ID NO:926).
In some cases, a MOD present in a TMMP is a CD80 polypeptide. In some cases, a CD80 polypeptide of a TMMP 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 following CD80 amino acid sequence: VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: (SEQ ID NO:927).
In some cases, a MOD present in a TMMP is a CD86 polypeptide. In some cases, a CD86 polypeptide of a TMMP 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 the following CD86 amino acid sequence:
In some cases, an immunomodulatory polypeptide present in a TMMP is an anti-CD28 antibody (e.g., an anti-CD28 scFv; an anti-CD28 nanobody; an anti-CD28 diabody; and the like). The ability of anti-CD28 antibodies to act as a superagonist, agonist, or antagonist of CD28 activity has been described. See e.g., Poirier et al., (2012) Amer. J. of Transplantation, “CD28-Specific Immunomodulating Antibodies: What Can Be Learned From Experimental Models?” 12:1682-1690. Of particular interest are anti-CD28 antibodies that act as an agonist or superagonist.
Thus, in some cases, a TMMP comprises an anti-CD28 antibody as at least one of the one or more immunomodulatory polypeptides. In some cases, a TMMP comprises, as the one or more immunomodulatory polypeptides: a) an anti-CD28 antibody; and b) one or more IL-2 polypeptides. In some cases, a TMMP comprises, as the one or more immunomodulatory polypeptides: a) an anti-CD28 antibody; and b) one or more IL-2 polypeptides, where the one or more IL-2 polypeptide are variant IL-2 polypeptides, as described above (e.g., an IL-2 polypeptide comprising H16A and F42A substitutions; an IL-2 polypeptide comprising H16T and F42A substitutions; an IL-2 polypeptide comprising H16T and F42T substitutions; and IL-2 polypeptide comprising H16A and F42T substitutions; and the like). In some cases, a TMMP comprises, as the one or more immunomodulatory polypeptides: a) an anti-CD28 antibody; and b) two copies of an IL-2 polypeptide comprising H16A and F42A substitutions. Examples of anti-CD28 VL and VH polypeptides are provided in below. In some cases, a TMMP comprises, as the one or more immunomodulatory polypeptides: a) an anti-CD28 antibody; and b) two copies of an IL-2 polypeptide comprising H16T and F42A substitutions. In some cases, a TMMP comprises, as the one or more immunomodulatory polypeptides: a) an anti-CD28 antibody; and b) one, two, or three copies of a 4-1BBL polypeptide. In some cases, a TMMP comprises, as the one or more immunomodulatory polypeptides: a) an anti-CD28 antibody; and b) three copies of a 4-1BBL polypeptide comprising a wild-type amino acid sequence. In some cases, a TMMP comprises, as the one or more immunomodulatory polypeptides: a) an anti-CD28 antibody; and b) three copies of a variant 4-1BBL polypeptide. The two different immunomodulatory polypeptides can be at any of a number of different positions in the first or the second polypeptide of the heterodimer of the TMMP. For example, in some cases, the anti-CD28 antibody is at the C-terminus of the first polypeptide; and the second immunomodulatory polypeptide (e.g., an IL-2 polypeptide (e.g., a variant IL-2 polypeptide); a 4-1BBL polypeptide; etc.) is at the N-terminus of the second polypeptide. As another example, in some cases, the anti-CD28 antibody is at the N-terminus of the second polypeptide; and the second immunomodulatory polypeptide (e.g., an IL-2 polypeptide (e.g., a variant IL-2 polypeptide); a 4-1BBL polypeptide; etc.) is at the C-terminus of the second polypeptide.
In some cases, an anti-CD28 antibody suitable for inclusion as an immunomodulatory polypeptide in a TMMP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: QWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYT FGGGTKLEIKR (SEQ ID NO:929); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSAL MSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTTVTVSS (SEQ ID NO:930). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987). In some cases, the VH CDRs are: DYGVH (SEQ ID NO:931) (VH CDR1); VIWAGGGTNYNSALMS (SEQ ID NO:932) (VH CDR2); and DKGYSYYYSMDY (SEQ ID NO:933) (VH CDR3).
In some cases, an anti-CD28 antibody suitable for inclusion as an immunomodulatory polypeptide in a TMMP comprises: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYT FGGGTKLEIKR (SEQ ID NO:929); and b) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-CD28 antibody suitable for inclusion as an immunomodulatory polypeptide in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYT FGGGTKLEIKR (SEQ ID NO:929); b) a peptide linker; and c) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSAL MSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTTVTVSS (SEQ ID NO:930). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-CD28 antibody suitable for inclusion as an immunomodulatory polypeptide in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSAL MSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTTVTVSS (SEQ ID NO:930); b) a peptide linker; and c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYT FGGGTKLEIKR (SEQ ID NO:929). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
An immunomodulatory polypeptide (i.e., one or more immunomodulatory polypeptides) can be present in a TMMP at any of a variety of positions. For example, an immunomodulatory polypeptide can be: 1)N-terminal to the MHC class I heavy chain (position 1); 2)C-terminal to the MHC class I heavy chain and N-terminal to the Ig Fc polypeptide; in other words, between the MHC class I heavy chain and the Ig Fc polypeptide (position 2); 3)C-terminal to the Ig Fc polypeptide (position 3); 4)N-terminal to the peptide epitope (position 4); or 5)C-terminal to the β2M polypeptide (position 5). Similarly, a TTP can be present in a TMMP at any of a variety of positions. Non-limiting examples of the various MOD positions and the various TTP positions are illustrated in
Of the embodiments illustrated in
A TMMP can comprise an Fc polypeptide or can comprise another suitable scaffold polypeptide.
Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds. Non-antibody-based scaffolds include, e.g., albumin, an XTEN (extended recombinant) polypeptide, transferrin, an Fc receptor polypeptide, an elastin-like polypeptide (see, e.g., Hassouneh et al. (2012) Methods Enzymol. 502:215; e.g., a polypeptide comprising a pentapeptide repeat unit of (Val-Pro-Gly-X-Gly; SEQ ID NO:935), where X is any amino acid other than proline), an albumin-binding polypeptide, a silk-like polypeptide (see, e.g., Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), a silk-elastin-like polypeptide (SELP; see, e.g., Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075), and the like. Suitable XTEN polypeptides include, e.g., those disclosed in WO 2009/023270, WO 2010/091122, WO 2007/103515, US 2010/0189682, and US 2009/0092582; see also Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitable albumin polypeptides include, e.g., human serum albumin.
Suitable scaffold polypeptides will in some cases be a half-life extending polypeptides. Thus, in some cases, a suitable scaffold polypeptide increases the in vivo half-life (e.g., the serum half-life) of the TMMP, compared to a control TMMP lacking the scaffold polypeptide. For example, in some cases, a scaffold polypeptide increases the in vivo half-life (e.g., the serum half-life) of the TMMP, compared to a control TMMP lacking the scaffold polypeptide, by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold. As an example, in some cases, an Fc polypeptide increases the in vivo half-life (e.g., the serum half-life) of the TMMP, compared to a control TMMP lacking the Fc polypeptide, by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold.
In some cases, a TMMP comprises an Ig Fc polypeptide. An Ig Fc polypeptide is also referred to herein as an “Fc polypeptide.” The Ig Fc polypeptide of a TMMP can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, etc., or a variant of a wild-type Ig Fc polypeptide. Variants include naturally occurring variants, non-naturally occurring variants, and combinations thereof. For example, the Ig Fc can be a variant of a Fc polypeptide such as a human IgG1 Fc, which variant has a substantially reduced ability to effect complement-dependent cytotoxicity (CDC) or antibody-dependent cell cytotoxicity (ADCC). See, e.g., the variant human IgG1 Fc polypeptides of
In some cases, the Fc polypeptide present in a TMMP comprises an amino acid sequence having at least 70%, 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 Fc amino acid sequence depicted in any one of
In some cases, the Fc polypeptide present in a TMMP is an IgG1 Fc polypeptide, or a variant of an IgG1 Fc polypeptide. For example, in some cases, the Fc polypeptide present in a TMMP comprises an amino acid sequence having at least 70%, 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 human IgG1 Fc polypeptide depicted in
In some cases, the Fc polypeptide present in a TMMP is an IgG1 Fc polypeptide, or a variant of an IgG1 Fc polypeptide, where variants include naturally occurring variants, non-naturally-occurring variants, and combinations thereof. For example, in some cases, the Fc polypeptide present in a TMMP comprises an amino acid sequence having at least 70%, 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 human IgG1 Fc polypeptide depicted in
In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in
In some cases, the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 human IgG2 Fc polypeptide depicted in
In some cases, the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 human IgG4 Fc polypeptide depicted in
In some cases, the IgG4 Fc polypeptide comprises the following amino acid sequence:
As discussed above, a TMMP can include one or more peptide linkers, i.e., a linker comprising a contiguous stretch of two or more amino acids, where the one or more linkers are between one or more components of a TMMP. For example, a TMMP can include one or more peptide linkers, i.e., a linker comprising a contiguous stretch of two or more amino acids, where the one or more linkers are between one or more of: i) an MHC class I heavy chain polypeptide and an Ig Fc polypeptide; ii) a MOD and an MHC class I polypeptide; iii) a first MOD and a second MOD; iv) a peptide and an MHC class I polypeptide; v) a peptide and a β2M polypeptide; vi) a TTP and an MHC class I heavy chain polypeptide; vii) a TTP and an Ig Fc polypeptide; and viii) a TTP and a MOD.
As used herein, the phrase “an optional peptide linker between any two of the components of a TMMP” refers to a peptide linker between any two adjacent polypeptides within the TMMP. For example, as used herein, the phrase “an optional peptide linker between any two of the components of a TMMP” refers to a peptide linker between one or more of:) an MHC class I heavy chain polypeptide and an Ig Fc polypeptide; ii) a MOD and an MHC class I polypeptide; iii) a first MOD and a second MOD; iv) a peptide and an MHC class I polypeptide; v) a peptide and a β2M polypeptide; vi) a TTP and an MHC class I heavy chain polypeptide; vii) a TTP and an Ig Fc polypeptide; and viii) a TTP and a MOD. As discussed below, linkers may be: a) a flexible peptide linker, including a short flexible peptide linker; or b) a rigid peptide linker.
Suitable linkers (also referred to as “spacers”) can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid to 25 amino acids, from 3 amino acids to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids. A suitable linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some cases, a linker has a length of from 25 amino acids to 50 amino acids, e.g., from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, or from 45 to 50 amino acids in length.
Exemplary flexible peptide linkers include glycine polymers (G)n (SEQ ID NO:946), glycine-serine polymers (including, for example, (GS)n (SEQ ID NO:937), (GSGGS)n (SEQ ID NO:938), (GGGGS)n (SEQ ID NO:934), and (GGGS)n (SEQ ID NO:939), where n is an integer of at least one and can be an integer from 1 to 10), glycine-alanine polymers, alanine-serine polymers, and other flexible peptide linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:940), GGSGG (SEQ ID NO:941), GSGSG (SEQ ID NO:942), GSGGG (SEQ ID NO:943), GGGSG (SEQ ID NO:944), GSSSG (SEQ ID NO:945), and the like.
Exemplary flexible peptide linkers include, e.g., (GGGGS)n (SEQ ID NO:934); also referred to as a “G4S” linker), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:947), where n is 2. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:875), where n is 3. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:876), where n is 4. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:948), where n is 7. In some cases, a linker comprises the amino acid sequence AAAGG (SEQ ID NO:874). Also suitable is a linker having the amino acid sequence AAAGG (SEQ ID NO:874). In TMMPs of this disclosure, the β2M polypeptide can be connected to the MHC heavy chain polypeptide by a (GGGGS)n (SEQ ID NO:934) linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., where n=3, n=4, or n=7.
As used in this disclosure, a “short flexible peptide linker” means a flexible peptide linker that comprises fewer than 15 amino acids, i.e., from 2-14 amino acids. For example, a short flexible peptide linker can comprise from 2-4 amino acids, from 2-5 amino acids, from 3-6 amino acids, from 4-8 amino acids, from 5-10 amino acids, or from 10-14 amino acids. Within this range includes flexible peptide linkers comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids.
In some cases, a peptide linker is a rigid peptide linker. As used herein, the term “rigid peptide linker” refers to a linker comprising a contiguous stretch of two or more amino acids that effectively separates protein domains by maintaining a substantially fixed distance/spatial separation between the domains, thereby reducing or substantially eliminating unfavorable interactions between such domains. For example, rigid linkers may be interposed when either the MOD(s) or the TTP are in Position 1 and/or in Position 3 discussed above. Rigid peptide linkers are known in the art and generally adopt a relatively well-defined conformation when in solution. Rigid peptide linkers include those which have a particular secondary and/or tertiary structure in solution; and are typically of a length sufficient to confer secondary or tertiary structure to the linker. Rigid peptide linkers include peptide linkers rich in proline, and peptide linkers having an inflexible helical structure, such as an α-helical structure. Rigid peptide linkers are described in, for example, Chen et al. (2013) Adv. Drug Deliv. Rev. 65:1357; and Klein et al. (2014) Protein Engineering, Design & Selection 27:325.
Examples of rigid peptide linkers include, e.g., (EAAAK)n (SEQ ID NO:949), A(EAAAK)nA (SEQ ID NO:950), A(EAAAK)nALEA(EAAAK)nA (SEQ ID NO:951), (Lys-Pro)n (SEQ ID NO:952), (Glu-Pro)n (SEQ ID NO:953), (Thr-Pro-Arg)n (SEQ ID NO:954), and (Ala-Pro)n (SEQ ID NO:955) where n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). Non-limiting examples of suitable rigid peptide linkers comprising EAAAK (SEQ ID NO:956) include EAAAK (SEQ ID NO:956), (EAAAK)2 (SEQ ID NO:958), (EAAAK)3 (SEQ ID NO:959), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO:960), and AEAAAKEAAAKA (SEQ ID NO:961). Non-limiting examples of suitable rigid peptide linkers comprising (AP)n include APAP (SEQ ID NO:962; also referred to herein as “(AP)2”); APAPAPAP (SEQ ID NO:963; also referred to herein as “(AP)4”); APAPAPAPAPAP (SEQ ID NO:964; also referred to herein as “(AP)6”); APAPAPAPAPAPAPAP (SEQ ID NO:965; also referred to herein as “(AP)8”); and APAPAPAPAPAPAPAPAPAP (SEQ ID NO:955; also referred to herein as “(AP)10”). Non-limiting examples of suitable rigid peptide linkers comprising (KP)n include KPKP (SEQ ID NO:967; also referred to herein as “(KP)2”); KPKPKPKP (SEQ ID NO:968; also referred to herein as “(KP)4”); KPKPKPKPKPKP (SEQ ID NO:969; also referred to herein as “(KP)6”); KPKPKPKPKPKPKPKP (SEQ ID NO:970; also referred to herein as “(KP)8”); and KPKPKPKPKPKPKPKPKPKP (SEQ ID NO:952; also referred to herein as “(KP)10”). Non-limiting examples of suitable rigid peptide linkers comprising (EP)n include EPEP (SEQ ID NO:972; also referred to herein as “(EP)2”); EPEPEPEP (SEQ ID NO:973; also referred to herein as “(EP)4”); EPEPEPEPEPEP (SEQ ID NO:974; also referred to herein as “(EP)6”); EPEPEPEPEPEPEPEP (SEQ ID NO:975; also referred to herein as “(EP)8”); and EPEPEPEPEPEPEPEPEPEP (SEQ ID NO:953; also referred to herein as “(EP)10”).
As noted above, in some cases, a linker peptide in a first polypeptide chain of a TMMP can include a cysteine residue that can form an interchain disulfide bond with a cysteine residue present in a second polypeptide chain. For example, as discussed above, in some cases a TMMP, or a dimerized TMMP such as a homodimer, comprises a linker between the peptide epitope and the β2M polypeptide that includes a cysteine residue that forms an interchain disulfide bond with a cysteine residue in the MHC class I heavy chain polypeptide present in the other chain of the TMMP. For example, in some cases, where a TMMP, or a dimerized TMMP such as a homodimer, comprises a cysteine-containing linker between the peptide epitope and the β2M polypeptide, the cysteine residue in the linker forms an interchain disulfide bond with a cysteine residue at amino acid 236 (e.g., formed by an A236C substitution) in the MHC class I heavy chain polypeptide present in the TMMP.
In some cases, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GCGGS (SEQ ID NO:977). In some cases, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:978), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; e.g., 1, 2, or 3. In some cases, the peptide linker between the peptide and the β2M polypeptide comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:882), where n is 2.
In some cases, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence CGGGS (SEQ ID NO:979). In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO:980), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; e.g., 1, 2, or 3.
In some cases, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GGCGS (SEQ ID NO:981). In some cases, the peptide linker comprises the amino acid sequence GGCGS(GGGGS)n (SEQ ID NO:982), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, e.g., 1, 2, or 3.
In some cases, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GGGCS (SEQ ID NO:983). In some cases, the peptide linker comprises the amino acid sequence GGGCS(GGGGS)n (SEQ ID NO:984), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.
In some cases, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GGGGC (SEQ ID NO:985). In some cases, the peptide linker comprises the amino acid sequence GGGGC(GGGGS)n (SEQ ID NO:986), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.
As discussed below, it has been found that the cytotoxic effect of TMMPs is greater when a TTP is positioned N-terminal to the MHC heavy chain polypeptide and the linker between them is no more than about 12 amino acids in length, e.g., no more than 10 amino acids in length (e.g., a (GGGGS)2 (SEQ ID NO:947) linker), no more than 8 amino acids in length, no more than 7 amino acids in length, no more than 6 amino acids in length, or no more than 5 amino acids in length, (e.g., a GGGGS (SEQ ID NO:873) linker). In some cases, the linker between them is no more than about 12 amino acids in length. In some cases, the linker between them is no more than 10 amino acids in length (e.g., a (GGGGS)2 (SEQ ID NO:947) linker). In some cases, the linker between them is no more than 8 amino acids in length. In some cases, the linker between them is no more than 7 amino acids in length. In some cases, the linker between them is no more than 6 amino acids in length. In some cases, the linker between them is no more than 5 amino acids in length, (e.g., a GGGGS (SEQ ID NO:873) linker).
As noted above, in some cases, a TMMP can form dimers. That is, the present disclosure provides a polypeptide comprising a dimer of two TMMPs. The present disclosure thus provides a protein that is a dimerized TMMP comprising two TMMPs that are covalently linked to each other. The covalent linkage of the dimer can be one or more disulfide bonds between an Ig Fc polypeptide in the first TMMP and an Ig Fc polypeptide in the second TMMP. As but one example, the Ig Fc can be a variant of a human IgG1 Fc polypeptide, which variant has a substantially reduced ability to effect complement-dependent cytotoxicity (CDC) or antibody-dependent cell cytotoxicity (ADCC) (e.g., the human IgG1 Fc polypeptide of
Accordingly, the present disclosure provides a protein comprising: a) a first TMMP; and b) a second TMMP, which optionally may be identical to the first TMMP, where the first and second TMMPs are covalently linked to one another. The covalent linkage can be a disulfide bond between an Ig Fc polypeptide in the first TMMP and an Ig Fc polypeptide in the second TMMP.
If desired, the Ig Fc polypeptides of each TMMP can comprise interspecific dimerization sequences, e.g., “Knob-in-Hole” sequences that permit two different TMMPs to selectively dimerize. Interspecific binding sequences favor formation of heterodimers with their cognate polypeptide sequence (i.e., the interspecific sequence and its counterpart interspecific sequence), particularly those based on Ig Fc sequence variants. Such interspecific polypeptide sequences include Knob-in-Hole, and Knob-in-Hole sequences that facilitate the formation of one or more disulfide bonds. For example, one interspecific binding pair comprises a T366Y and Y407T mutant pair in the CH3 domain interface of IgG1, or the corresponding residues of other immunoglobulins. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996). A second interspecific binding pair involves the formation of a knob by a T366W substitution, and a hole by the triple substitutions T366S, L368A and Y407V on the complementary Ig Fc sequence. See Xu et al. mAbs 7:1, 231-242 (2015). Another interspecific binding pair has a first Fc polypeptide with Y349C, T366S, L368A, and Y407V substitutions and a second Ig Fc polypeptide with S354C, and T366W substitutions (disulfide bonds can form between the Y349C and the S354C). See, e.g., Brinkmann and Konthermann, mAbs 9:2, 182-212 (2015). Ig Fc polypeptide sequences, either with or without knob-in-hole modifications, can be stabilized by the formation of disulfide bonds between the Ig Fc polypeptides (e.g., the hinge region disulfide bonds). Thus, in some cases, a dimerized TMMP can be a heterodimer, comprising two TMMP chains that are not identical in amino acid sequence.
Interspecific dimerization sequences also may be employed to enable TMMPs to be linked to non-TMMP molecules that can provide additional functionality to the TMMP. For example, a TMMP could be linked to a molecule that comprise polypeptides (e.g., antibodies or binding fragments thereof such as scFvs) that bind to cancer-associated antigens, thereby enabling the TMMP to localize to tissues comprising the cancer-associated antigen.
As discussed above, a TMMP includes, in the first and/or the second polypeptide, a tumor-targeting polypeptide (TTP), i.e., a polypeptide specific for a cancer-associated epitope. A “cancer-associated” epitope is an epitope that is present in a cancer-associated antigen. In some cases, a TTP is an antibody. In some cases, a TTP is a single-chain T-cell receptor (scTCR).
In some cases, a TTP present in a TMMP targets a cancer-associated antigen. In some cases, the target of a TTP is a peptide/HLA (pHLA) complex on the surface of a cancer cell, where the peptide can be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen).
Cancer-associated antigens that can be targeted with a tumor-targeting polypeptide present in a TMMP include, e.g., NY-ESO (New York Esophageal Squamous Cell Carcinoma 1), MART-1 (melanoma antigen recognized by T cells 1, also known as Melan-A), HPV (human papilloma virus) E6, BCMA (B-cell maturation antigen), CD123, CD133, CD171, CD19, CD20, CD22, CD30, CD33, CD38, CD138, CEA (carcinoembryonic antigen), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor variant III), EpCAM (epithelial cell adhesion molecule), EphA2 (ephrin type-A receptor 2), disialoganglioside GD2, GPC3 (glypican-3), HER2, IL13Ralpha2 (Interleukin 13 receptor subunit alpha-2), LeY (a difucosylated type 2 blood group-related antigen), MAGE-A3 (melanoma-associated antigen 3), melanoma glycoprotein, mesothelin, MUC1 (mucin 1), MUC16 (mucin-16), myelin, NKG2D (Natural Killer Group 2D) ligands, PSMA (prostate specific membrane antigen), and RORI (type I receptor tyrosine kinase-like orphan receptor).
Cancer-associated antigens that can be targeted with a TTP present in a TMMP include, but are not limited to, 17-1A-antigen, alpha-fetoprotein (AFP), alpha-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE, bcl-2, bcl-6, BCMA, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX (CAIX), CASP-8/m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD138, CD147, CD154, CD171, CDC27, CDK-4/m, CDKN2A, CEA, CEACAM5, CEACAM6, claudin (e.g., claudin-1, claudin-10, claudin-18 (e.g., claudin-18, isoform 2)), complement factors (such as C3, C3a, C3b, C5a and C5), colon-specific antigen-p (CSAp), c-Met, CTLA-4, CXCR4, CXCR7, CXCL12, DAM, Dickkopf-related protein (DKK), ED-B fibronectin, epidermal growth factor receptor (EGFR), EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, EphA2, EphA3, fibroblast activation protein (FAP), fibroblast growth factor (FGF), Flt-1, Flt-3, folate binding protein, folate receptor, G250 antigen, gangliosides (such as GC2, GD3 and GM2), GAGE, GD2, gp100, GPC3, GRO-13, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2, HER3, HMGB-1, hypoxia inducible factor (HIF-1), HIF-1a, HSP70-2M, HST-2, Ia, IFN-gamma, IFN-alpha, IFN-beta, IFN-X, IL-4R, IL-6R, IL-13R, IL13Ralpha2, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, ILGF, ILGF-1R, insulin-like growth factor-1 (IGF-1), IGF-1R, integrin aV 3, integrin a51, KC4-antigen, killer-cell immunoglobulin-like receptor (KIR), Kras, KS-1-antigen, KS1-4, LDR/FUT, Legamma, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, mCRP, MCP-1, melanoma glycoprotein, mesothelin, MIP-1A, MIP-1B, MIF, mucins (such as MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2 and MUM-3), NCA66, NCA95, NCA90, Nectin-4, NY-ESO-1, PAM4 antigen, pancreatic cancer mucin, PD-1, PD-L1, PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, RSS, RANTES, SAGE, 5100, survivin, survivin-2B, T101, TAC, TAG-72, tenascin, Thomson-Friedenreich antigens, Tn antigen, TNF-alpha, tumor necrosis antigens, TRAG-3, TRAIL receptors, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR) and WT-1.
In some cases, the cancer-associated antigen is an antigen associated with a hematological cancer. Examples of such antigens include, but are not limited to, BCMA, C5, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD56, CD66, CD74, CD79a, CD79b, CD80, CD138, CTLA-4, CXCR4, DKK, EphA3, GM2, HLA-DR beta, integrin αVβ3, IGF-R1, IL6, KIR, PD-1, PD-L1, TRAILR1, TRAILR2, transferrin receptor, and VEGF. In some cases, the cancer-associated antigen is an antigen expressed by malignant B cells, such as CD19, CD20, CD22, CD25, CD38, CD40, CD45, CD74, CD80, CTLA-4, IGF-R1, IL6, PD-1, TRAILR2, or VEGF.
In some cases, the cancer-associated antigen is an antigen associated with a solid tumor. Examples of such antigens include, but are not limited to, CAIX, cadherins, CEA, c-MET, CTLA-4, EGFR family members, EpCAM, EphA3, FAP, folate-binding protein, FR-alpha, gangliosides (such as GC2, GD3 and GM2), HER2, HER3, IGF-1R, integrin αVβ3, integrin α5β1, Legamma, Liv1, mesothelin, mucins, NaPi2b, PD-1, PD-L1, PD-1 receptor, pgA33, PSMA, RANKL, RORI, TAG-72, tenascin, TRAILR1, TRAILR2, VEGF, VEGFR, and others listed above.
In some cases, the target of a TTP is a peptide/HLA (pHLA) complex on the surface of a cancer cell, where the peptide can be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen). Cancer-associated peptides are known in the art. In some cases, a cancer-associated peptide is bound to an HLA complex comprising an HLA-A*0201 heavy chain and a β2M polypeptide.
In some cases, the epitope present in the pHLA on the surface of a cancer cell is bound to an HLA complex comprising an HLA heavy chain such as HLA-A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and/or A*3401. In some cases, the epitope present in the pHLA on the surface of a cancer cell is bound to an HLA complex comprising an HLA heavy chain such as HLA-B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and/or B*5301. In some cases, the epitope present in the pHLA on the surface of a cancer cell is bound to an HLA complex comprising an HLA heavy chain such as C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*702, C*0801, and/or C*1502.
In some cases, the epitope is a cancer-associated epitope of any one of the following cancer-associated antigens: a MUC1 polypeptide, an LMP2 polypeptide, an epidermal growth factor receptor (EGFR) vIII polypeptide, a HER-2/neu polypeptide, a melanoma antigen family A, 3 (MAGE A3) polypeptide, a p53 polypeptide, a mutant p53 polypeptide, an NY-ESO-1 polypeptide, a folate hydrolase (prostate-specific membrane antigen; PSMA) polypeptide, a carcinoembryonic antigen (CEA) polypeptide, a claudin polypeptide (e.g., claudin-1, claudin-10, claudin-18 (e.g., claudin-18, isoform 2)), a Nectin-4 polypeptide, a melanoma antigen recognized by T-cells (melanA/MART1) polypeptide, a Ras polypeptide, a gp100 polypeptide, a proteinase3 (PR1) polypeptide, a bcr-abl polypeptide, a tyrosinase polypeptide, a survivin polypeptide, a prostate specific antigen (PSA) polypeptide, an hTERT polypeptide, a sarcoma translocation breakpoints polypeptide, a synovial sarcoma X (SSX) breakpoint polypeptide, an EphA2 polypeptide, an acid phosphatase, prostate (PAP) polypeptide, a melanoma inhibitor of apoptosis (ML-IAP) polypeptide, an epithelial cell adhesion molecule (EpCAM) polypeptide, an ERG (TMPRSS2 ETS fusion) polypeptide, a NA17 polypeptide, a paired-box-3 (PAX3) polypeptide, an anaplastic lymphoma kinase (ALK) polypeptide, an androgen receptor polypeptide, a cyclin B1 polypeptide, an N-myc proto-oncogene (MYCN) polypeptide, a Ras homolog gene family member C (RhoC) polypeptide, a tyrosinase-related protein-2 (TRP-2) polypeptide, a mesothelin polypeptide, a prostate stem cell antigen (PSCA) polypeptide, a melanoma associated antigen-1 (MAGE A1) polypeptide, a cytochrome P450 1B1 (CYPIB1) polypeptide, a placenta-specific protein 1 (PLAC1) polypeptide, a BORIS polypeptide (also known as CCCTC-binding factor or CTCF), an ETV6-AML polypeptide, a breast cancer antigen NY-BR-1 polypeptide (also referred to as ankyrin repeat domain-containing protein 30A), a regulator of G-protein signaling (RGS5) polypeptide, a squamous cell carcinoma antigen recognized by T-cells (SART3) polypeptide, a carbonic anhydrase IX polypeptide, a paired box-5 (PAX5) polypeptide, an OY-TES1 (testis antigen; also known as acrosin binding protein) polypeptide, a sperm protein 17 polypeptide, a lymphocyte cell-specific protein-tyrosine kinase (LCK) polypeptide, a high molecular weight melanoma associated antigen (HMW-MAA), an A-kinase anchoring protein-4 (AKAP-4), a synovial sarcoma X breakpoint 2 (SSX2) polypeptide, an X antigen family member 1 (XAGE1) polypeptide, a B7 homolog 3 (B7H3; also known as CD276) polypeptide, a legumain polypeptide (LGMN1; also known as asparaginyl endopeptidase), a tyrosine kinase with Ig and EGF homology domains-2 (Tie-2; also known as angiopoietin-1 receptor) polypeptide, a P antigen family member 4 (PAGE4) polypeptide, a vascular endothelial growth factor receptor 2 (VEGF2) polypeptide, a MAD-CT-1 polypeptide, a fibroblast activation protein (FAP) polypeptide, a platelet derived growth factor receptor beta (PDGFO) polypeptide, a MAD-CT-2 polypeptide, a Fos-related antigen-1 (FOSL) polypeptide; a human papilloma virus (HPV) antigen; an alpha-feto protein (AFP) antigen; and a Wilms tumor-1 (WTi) antigen.
For example, in some cases, a TTP present in a TMMP binds to: a) a WT-1 peptide bound to an HLA complex comprising an HLA heavy chain (e.g., an HLA-A*0201 heavy chain or an HLA-A*2402 heavy chain) and a β2M polypeptide; b) an HPV peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; c) a mesothelin peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; d) a Her2 peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; or e) a BCMA peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide.
In some cases, a cancer-associated peptide is a peptide of a mesothelin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following mesothelin amino acid sequence:
In some cases, a cancer-associated peptide is a peptide of a Her2 polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Her2 (receptor tyrosine-protein kinase erbB2) amino acid sequence:
In some cases, a cancer-associated peptide is a peptide of a BCMA polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following BCMA amino acid sequence:
In some cases, a cancer-associated peptide is a peptide of a WT-1 polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following WT-1 amino acid sequence:
Non-limiting examples of WT-1 peptides include RMFPNAPYL (SEQ ID NO:1002), CMTWNQMN (SEQ ID NO:1003), CYTWNQMNL (SEQ ID NO:1004), CMTWNQMNLGATLKG (SEQ ID NO:1005), WNQMNLGATLKGVAA (SEQ ID NO:1006), CMTWNYMNLGATLKG (SEQ ID NO:1007), WNYMNLGATLKGVAA (SEQ ID NO:1008), MTWNQMNLGATLKGV (SEQ ID NO:1009), TWNQMNLGATLKGVA (SEQ ID NO:1010), CMTWNLMNLGATLKG (SEQ ID NO:1011), MTWNLMNLGATLKGV (SEQ ID NO:1012), TWNLMNLGATLKGVA (SEQ ID NO:1013), WNLMNLGATLKGVAA (SEQ ID NO:1014), MNLGATLK (SEQ ID NO:1015), MTWNYMNLGATLKGV (SEQ ID NO:1016), TWNYMNLGATLKGVA (SEQ ID NO:1017), CMTWNQMNLGATLKGVA (SEQ ID NO:1018), CMTWNLMNLGATLKGVA (SEQ ID NO:1019), CMTWNYMNLGATLKGVA (SEQ ID NO:1020), GYLRNPTAC (SEQ ID NO:1021), GALRNPTAL (SEQ ID NO:1022), YALRNPTAC (SEQ ID NO:1023), GLLRNPTAC (SEQ ID NO:1024), RYRPHPGAL (SEQ ID NO:1025), YQRPHPGAL (SEQ ID NO:1026), RLRPHPGAL (SEQ ID NO:1027), RIRPHPGAL (SEQ ID NO:1028), QFPNHSFKHEDPMGQ (SEQ ID NO:1029), HSFKHEDPY (SEQ ID NO:1030), QFPNHSFKHEDPM (SEQ ID NO:1031), QFPNHSFKHEDPY (SEQ ID NO:1032), KRPFMCAYPGCNK (SEQ ID NO:1033), KRPFMCAYPGCYK (SEQ ID NO:1034), FMCAYPGCY (SEQ ID NO:1035), FMCAYPGCK (SEQ ID NO:1036), KRPFMCAYPGCNKRY (SEQ ID NO:1037), SEKRPFMCAYPGCNK (SEQ ID NO:1038), KRPFMCAYPGCYKRY (SEQ ID NO:1039), NLMNLGATL (SEQ ID NO:1040), and NYMNLGATL (SEQ ID NO:1041).
In some cases, a cancer-associated peptide is a peptide of a human papillomavirus (HPV) polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to an HPV polypeptide. An HPV peptide can be a peptide of an HPV E6 polypeptide or an HPV E7 polypeptide. The HPV epitope can be an epitope of HPV of any of a variety of genotypes, including, e.g., HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, or HPV82. Non-limiting examples of HPV peptides include: E6 18-26 (KLPQLCTEL; SEQ ID NO:1042); E6 26-34 (LQTTIHDII; SEQ ID NO:1043); E6 49-57 (VYDFAFRDL; SEQ ID NO:1044); E6 52-60 (FAFRDLCIV; SEQ ID NO:1045); E6 75-83 (KFYSKISEY; SEQ ID NO:1046); E6 80-88 (ISEYRHYCY; SEQ ID NO:1047); E7 7-15 (TLHEYMLDL; SEQ ID NO:1048); E7 11-19 (YMLDLQPET; SEQ ID NO:1049); E7 44-52 (QAEPDRAHY; SEQ ID NO:1050); E7 49-57 (RAHYNIVTF (SEQ ID NO:1051); E7 61-69 (CDSTLRLCV; SEQ ID NO:1052); and E7 67-76 (LCVQSTHVDI; SEQ ID NO:1053); E7 82-90 (LLMGTLGIV; SEQ ID NO:1054); E7 86-93 (TLGIVCPI; SEQ ID NO:1055); and E7 92-93 (LLMGTLGIVCPI; SEQ ID NO:1056).
In some cases, a cancer-associated peptide is a peptide of a claudin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following claudin-18 (isoform 2) (CLDN 18.2) amino acid sequence:
In some cases, a cancer-associated peptide is a peptide of a trophoblast cell-surface antigen-2 (Trop-2) polypeptide. Trop-2 (also known as epithelial glycoprotein-1, gastrointestinal tumor-associated antigen GA733-1, membrane component chromosome 1 surface marker-1, and tumor-associated calcium signal transducer-2) is a transmembrane glycoprotein that is upregulated in numerous cancer types, and is the protein product of the TACSTD2 gene. In some cases, a cancer-associated peptide is a peptide of a TROP-2 polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following TROP-2 amino acid sequence:
As noted above, in some cases, a TTP present in a TMMP is an antibody. In some cases, the TTP is an antibody that is specific for a cancer-associated antigen. In some cases, the TTP is an antibody specific for a peptide/HLA complex on the surface of a cancer cell, where the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).
Non-limiting examples of cancer-associated antigen-targeted antibodies that can be included in a TMMP include, but are not limited to, abituzumab (anti-CD51), LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), binutuzu (anti-CD20), binutuzumab (GA101, anti-CD20), daratumumab (anti-CD38), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-TROP-2), PAM4 or KC4 (both anti-mucin), MN-14 (anti-CEA), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu 31 (anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), oportuzumab (anti-EpCAM), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20); PAM4 (also known as clivatuzumab; anti-mucin), trastuzumab (anti-HER2), pertuzumab (anti-HER2), polatuzumab (anti-CD79b), and anetumab (anti-mesothelin).
In some cases, the tumor-targeting polypeptide is a single-chain antibody. In some cases, the tumor-targeting polypeptide is a scFv. In some cases, the tumor-targeting polypeptide is a nanobody (also referred to as a single domain antibody (sdAb)). In some cases, the tumor-targeting polypeptide is a heavy chain nanobody. In some cases, the tumor-targeting polypeptide is a light chain nanobody.
VH and VL amino acid sequences of various tumor antigen-binding antibodies are known in the art, as are the light chain and heavy chain CDRs of such antibodies. See, e.g., Ling et al. (2018) Frontiers Immunol. 9:469; WO 2005/012493; US 2019/0119375; US 2013/0066055. The following are non-limiting examples of tumor antigen-binding antibodies.
In some cases, an anti-Her2 antibody comprises: a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-Her2 antibody comprises a light chain variable region (VL) present in the light chain amino acid sequence provided above; and a heavy chain variable region (VH) present in the heavy chain amino acid sequence provided above. For example, an anti-Her2 antibody can comprise: a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS RSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:1063); and b) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:1064). In some cases, an anti-Her2 antibody comprises, in order from N-terminus to C-terminus: a) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:1065); b) a linker; and c) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS RSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:1066). Suitable linkers are described elsewhere herein and include, e.g., (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
In some cases, an anti-Her2 antibody comprises VL CDR1, VL CDR2, and VL CDR3 present in the light chain amino acid sequence provided above; and VH CDR1, CDR2, and CDR3 present in the heavy chain amino acid sequence provided above. In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987).
For example, an anti-Her2 antibody can comprise a VL CDR1 having the amino acid sequence RASQDVNTAVA (SEQ ID NO:1067); a VL CDR2 having the amino acid sequence SASFLY (SEQ ID NO:1068); a VL CDR3 having the amino acid sequence QQHYTTPP (SEQ ID NO:1069); a VH CDR1 having the amino acid sequence GFNIKDTY (SEQ ID NO:1070); a VH CDR2 having the amino acid sequence IYPTNGYT (SEQ ID NO:1071); and a VH CDR3 having the amino acid sequence SRWGGDGFYAMDY (SEQ ID NO:1072).
In some cases, an anti-Her2 antibody is a scFv antibody. For example, an anti-Her2 scFv can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
As another example, in some cases, an anti-Her2 antibody comprises: a) a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-Her2 antibody comprises a VL present in the light chain amino acid sequence provided above; and a VH present in the heavy chain amino acid sequence provided above. For example, an anti-Her2 antibody can comprise: a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO:1076); and b) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence:
In some cases, an anti-Her2 antibody comprises VL CDR1, VL CDR2, and VL CDR3 present in the light chain amino acid sequence provided above; and VH CDR1, CDR2, and CDR3 present in the heavy chain amino acid sequence provided above. In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987).
For example, an anti-HER2 antibody can comprise a VL CDR1 having the amino acid sequence KASQDVSIGVA (SEQ ID NO:1078); a VL CDR2 having the amino acid sequence SASYRY (SEQ ID NO:1079); a VL CDR3 having the amino acid sequence QQYYIYPY (SEQ ID NO:1080); a VH CDR1 having the amino acid sequence GFTFTDYTMD (SEQ ID NO:1081); a VH CDR2 having the amino acid sequence ADVNPNSGGSIYNQRFKG (SEQ ID NO:1082); and a VH CDR3 having the amino acid sequence ARNLGPSFYFDY (SEQ ID NO:1083).
In some cases, an anti-Her2 antibody is a scFv. For example, in some cases, an anti-Her2 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
Anti-CD19 antibodies are known in the art; and the VH and VL, or the VH and VL CDRs, of any anti-CD19 antibody can be used in a TMMP. See e.g., WO 2005/012493.
In some cases, an anti-CD19 antibody includes a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO:1084); a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO:1085); and a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO:1086). In some cases, an anti-CD19 antibody includes a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO:1087); a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO:1088); and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO:1089). In some cases, an anti-CD19 antibody includes a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO:1084); a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO:1085); a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO:1086); a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO:1087); a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO:1088); and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO:1089).
In some cases, an anti-CD19 antibody is a scFv. For example, in some cases, an anti-CD19 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
Anti-mesothelin antibodies are known in the art; and the VH and VL, or the VH and VL CDRs, of any anti-mesothelin antibody can be used in a TMMP. See, e.g., U.S. 2019/0000944; WO 2009/045957; WO 2014/031476; U.S. Pat. No. 8,460,660; US 2013/0066055; and WO 2009/068204. In some cases, the TTP is an anti-mesothelin scFv or an anti-mesothelin nanobody comprising VH and VL CDRs present in any one of the amino acid sequences set forth in
In some cases, an anti-mesothelin antibody comprises: a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNR PSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS (SEQ ID NO:1091); and b) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-mesothelin antibody comprises a VL present in the light chain amino acid sequence provided above; and a VH present in the heavy chain amino acid sequence provided above. For example, an anti-mesothelin antibody can comprise: a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFS GSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTK (SEQ ID NO:1093); and b) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence:
In some cases, an anti-mesothelin antibody comprises VL CDR1, VL CDR2, and VL CDR3 present in the light chain amino acid sequence provided above; and VH CDR1, CDR2, and CDR3 present in the heavy chain amino acid sequence provided above. In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987).
For example, an anti-mesothelin antibody can comprise a VL CDR1 having the amino acid sequence TGTSSDIGGYNSVS (SEQ ID NO:1095); a VL CDR2 having the amino acid sequence LMIYGVNNRPS (SEQ ID NO:1096); a VL CDR3 having the amino acid sequence SSYDIESATP (SEQ ID NO:1097); a VH CDR1 having the amino acid sequence GYSFTSYWIG (SEQ ID NO:1098); a VH CDR2 having the amino acid sequence WMGIIDPGDSRTRYSP (SEQ ID NO:1099); and a VH CDR3 having the amino acid sequence GQLYGGTYMDG (SEQ ID NO:1100).
An anti-mesothelin antibody can be a scFv. As one non-limiting example, an anti-mesothelin scFv can comprise the following amino acid sequence:
INPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARGR
YYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSL
where VH CDR1, CDR2, and CDR3 are underlined; and VL CDR1, CDR2, and CDR3 are bolded and underlined.
As one non-limiting example, an anti-mesothelin scFv can comprise the following amino acid sequence:
INPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDL
RRTVVTPRAYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQL
ET
GVPSRFSGSGSGTDFSFTISSLQPEDIATYYCQQHDNLPLTFGQGTKV
where VH CDR1, CDR2, and CDR3 are underlined; and VL CDR1, CDR2, and CDR3 are bolded and underlined.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO:1103); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYV DSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO:1104). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987). See, e.g., BMS6A5.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP comprises: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO:1103); and b) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYV DSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO:1104); b) a peptide linker; and c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO:1103). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO:1103); b) a peptide linker; and c) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYV DSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO:1104). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGS GSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO:1105); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKF RGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO:1106). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987). See, e.g., Amatuximab.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP comprises: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGS GSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO:1105); and b) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGS GSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO:1105); b) a peptide linker; and c) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKF RGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO:1106). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKF RGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO:1106); b) a peptide linker; and c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGS GSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO:1105). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFS GSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO:1107); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSF QGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO:1094). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987).
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP comprises: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFS GSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO:1107); and b) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFS GSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO:1107); b) a peptide linker; and c) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSF QGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO:1094). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSF QGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO:1094); b) a peptide linker; and c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFS GSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO:1107). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO:1108); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYN QKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO:1109). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987). See, e.g., RG7787.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP comprises: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO:1108); and b) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO:1108); b) a peptide linker; and c) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYN QKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO:1109). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYN QKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO:1109); b) a peptide linker; and c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO:1108). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-mesothelin scFv comprises the following amino acid sequence:
QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAV
IWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREG
DGSGIYYYYGMDVWGQGTTVTVSS
GGGGSGGGGSGGGGSEIVLTQSPGTL
SLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD
RESGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK,
the VH sequence is italicized, the (GGGGS)3 (SEQ ID NO:875) linker is bolded and underlined, and the VL sequence is underlined.
In some cases, an anti-mesothelin scFv comprises the following amino acid sequence:
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY
GASSRATGIPDRESGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTF
GPGTKVDIKGGGGSGGGGSGGGGS
QMQLVESGGGVVQPGRSLRLSCTASG
FTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSK
NTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS,
where the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bolded and underlined, and the VL sequence is italicized.
In some cases, an anti-mesothelin scFv comprises the following amino acid sequence:
QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGL
ITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGG
YDGRGFDYWGSGTPVTVSS
GGGGSGGGGSGGGGSDIELTOSPAIMSASPG
EKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRESGSGS
GNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK,
where the VH sequence is italicized, the (GGGGS)3 (SEQ ID NO:875) linker is bolded and underlined, and the VL sequence is underlined.
In some cases, an anti-mesothelin scFv comprises the following amino acid sequence:
DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTS
KLASGVPGRESGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTK
VEIKGGGGSGGGGSGGGGS
QVQLQQSGPELEKPGASVKISCKASGYSFTGY
TMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDL
LSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS,
where the V L sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bolded and underlined, and the VL sequence is italicized.
In some cases, an anti-mesothelin scFv comprises the following amino acid sequence:
QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGII
DPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLY
GGTYMDGWGQGTLVTVSS
GGGGSGGGGSGGGGSDIALTQPASVSGSPGQSI
TISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKS
GNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG,
where the VH sequence is italicized, the (GGGGS)3 (SEQ ID NO:875) linker is bolded and underlined, and the VL sequence is underlined.
In some cases, an anti-mesothelin scFv comprises the following amino acid sequence:
DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIY
GVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVF
GGGTKLTVLGGGGGSGGGGSGGGGS
QVELVQSGAEVKKPGESLKISCKGSG
YSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSIS
TAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS,
where the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bolded and underlined, and the VL sequence is italicized.
In some cases, an anti-mesothelin scFv comprises the following amino acid sequence:
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLI
TPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYD
GRGFDYWGQGTLVTVSS
GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRV
TITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRESGSGSGTDF
TLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK,
where the VH sequence is italicized, the (GGGGS)3 (SEQ ID NO:875) linker is bolded and underlined, and the VL sequence is underlined.
In some cases, an anti-mesothelin scFv comprises the following amino acid sequence:
DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTS
KLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTK
LEIKGGGGSGGGGSGGGGS
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGY
TMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMEL
SSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS,
where the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bolded and underlined, and the VL sequence is italicized.
Prostate-specific membrane antigen (PSMA) (also known as folate hydrolase 1 (FOLH1); membrane glutamate carboxypeptidase, and N-Acetylated-Alpha-Linked Acidic Dipeptidase 1) that is up-regulated in cancerous cells in the prostate and is used as a diagnostic and prognostic indicator of prostate cancer.
Anti-PSMA antibodies are known in the art; and the VH and VL, or the VH and VL CDRs, of any anti-PSMA antibody can be used in a TMMP. See, e.g., U.S. Pat. No. 10,179,819 and U.S. Patent Publication No. 2021/0277141.
CD22 (also known as B-Lymphocyte Cell Adhesion Molecule, Sialic Acid-Binding Ig-Like Lectin 2, or SIGLEC2) is a sialic acid-binding adhesion molecule largely restricted to the B cell lineage and expressed on most B-lineage malignancies.
Anti-CD22 antibodies are known in the art; and the VH and VL, or the VH and VL CDRs, of any anti-CD22 antibody can be used in a TMMP. See, e.g., Xiao et al. (2009) Mabs 1:297 (describing the fully human anti-CD22 m971 scFv); and U.S. Patent Publication No. 2020/0147134. Examples of anti-CD22 antibodies include epratuzumab and inotuzumab. See, e.g., Lenoard et al. (2007) Oncogene 26:3704 and U.S. Pat. No. 5,789,554 (describing epratuzumab); and DiJoseph et al. (2007) Leukemia 21:2240 (describing inotuzumab).
For example, an anti-CD22 antibody can comprise: i) a heavy chain variable region (VH) CDR1 having the amino acid sequence: GDSVSSNSAA (SEQ ID NO:1110); ii) a VH CDR2 having the amino acid sequence: TYYRSKWYN (SEQ ID NO:1111); iii) a VH CDR3 having the amino acid sequence: AREVTGDLEDAFDI (SEQ ID NO:1112); iv) a light chain variable region (VL) CDR1 having the amino acid sequence: QTIWSY (SEQ ID NO:1113); v) a VL CDR2 having the amino acid sequence: AAS (Ala-Ala-Ser); and vi) a VL CDR3 having the amino acid sequence: QQSYSIPQT (SEQ ID NO:1114).
Trophoblast cell surface antigen 2 (Trop-2) (also known as epithelial glycoprotein-1, gastrointestinal tumor-associated antigen GA733-1, membrane component chromosome 1 surface marker-1, and tumor-associated calcium signal transducer-2) is a transmembrane glycoprotein that is upregulated in numerous cancer types, and is the protein product of the TACSTD2 gene.
In some cases, the TTP is an anti-TROP-2 scFv or an anti-TROP-2 nanobody comprising VH and VL CDRs present in any one of the amino acid sequences set forth in
Anti-TROP-2 antibodies are known in the art; and the VH and VL, or the VH and VL CDRs, of any anti-TROP-2 antibody can be used in a TMMP. See, e.g., U.S. Pat. No. 7,238,785). In some cases, an anti-TROP-2 antibody comprises: i) light chain CDR sequences CDR1 (KASQDVSIAVA; SEQ ID NO:1115); CDR2 (SASYRYT; SEQ ID NO:1116); and CDR3 (QQHYITPLT; SEQ ID NO:1117); and ii) heavy chain CDR sequences CDR1 (NYGMN; SEQ ID NO:1118); CDR2 (WINTYTGEPTYTDDFKG; SEQ ID NO:1119); and CDR3 (GGFGSSYWYFDV; SEQ ID NO:1120).
In some cases, an anti-TROP-2 antibody comprises: i) heavy chain CDR sequences CDR1 (TAGMQ; SEQ ID NO:1121); CDR2 (WINTHSGVPKYAEDFKG (SEQ ID NO:1122); and CDR3 (SGFGSSYWYFDV; SEQ ID NO:1123); and ii) light chain CDR sequences CDR1 (KASQDVSTAVA; SEQ ID NO:1124); CDR2 (SASYRYT; SEQ ID NO:1116); and CDR3 (QQHYITPLT; SEQ ID NO:1117).
In some cases, an anti-TROP2 antibody suitable for inclusion in a TMMP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGS GSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO:1125); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYT DDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO:1126). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987).
In some cases, an anti-TROP-2 antibody suitable for inclusion in a TMMP comprises: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGS GSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO:1125); and b) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-TROP-2 antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGS GSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO:1125); b) a peptide linker; and c) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYT DDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO:1126). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-TROP-2 antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYT DDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO:1126); b) a peptide linker; and c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGS GSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO:1125). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-TROP2 antibody suitable for inclusion in a TMMP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSG SGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO:1127); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYA EDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO:1128). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987).
In some cases, an anti-TROP-2 antibody suitable for inclusion in a TMMP comprises: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSG SGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO:1127); and b) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-TROP-2 antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSG SGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO:1127); b) a peptide linker; and c) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYA EDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO:1128). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
In some cases, an anti-TROP-2 antibody suitable for inclusion in a TMMP is a scFv comprising, in order from N-terminus to C-terminus: a) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYA EDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO:1128); b) a peptide linker; and c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSG SGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO:1127). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:875) and has a length of 15 amino acids.
Anti-BCMA (B-cell maturation antigen) antibodies are known in the art; and the VH and VL, or the VH and VL CDRs, of any anti-BCMA antibody can be used in a TMMP. See, e.g., WO 2014/089335; US 2019/0153061; and WO 2017/093942.
In some cases, an anti-BCMA antibody comprises: a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-BCMA antibody comprises a VL present in the light chain amino acid sequence provided above; and a VH present in the heavy chain amino acid sequence provided above. For example, an anti-BCMA antibody can comprise: a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence:
In some cases, an anti-BCMA antibody comprises VL CDR1, VL CDR2, and VL CDR3 present in the light chain amino acid sequence provided above; and VH CDR1, CDR2, and CDR3 present in the heavy chain amino acid sequence provided above. In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Table 1, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Table 1, above; and Chothia 1987).
For example, an anti-BCMA antibody can comprise a VL CDR1 having the amino acid sequence SSNIGSNT (SEQ ID NO:1133), a VL CDR2 having the amino acid sequence NYH, a VL CDR3 having the amino acid sequence AAWDDSLNGWV (SEQ ID NO:1134)), a VH CDR1 having the amino acid sequence GFTFGDYA (SEQ ID NO: 1135), a VH CDR2 having the amino acid sequence SRSKAYGGTT (SEQ ID NO:1136), and a VH CDR3 having the amino acid sequence ASSGYSSGWTPFDY (SEQ ID NO:1137).
An anti-BCMA antibody can be a scFv. As one non-limiting example, an anti-BCMA scFv can comprise the following amino acid sequence:
As another example, an anti-BCMA scFv can comprise the following amino acid sequence:
In some cases, an anti-BCMA antibody can comprise a VL CDR1 having the amino acid sequence SASQDISNYLN (SEQ ID NO:1140); a VL CDR2 having the amino acid sequence YTSNLHS (SEQ ID NO:1141); a VL CDR3 having the amino acid sequence QQYRKLPWT (SEQ ID NO:1142); a VH CDR1 having the amino acid sequence NYWMH (SEQ ID NO:1143); a VH CDR2 having the amino acid sequence ATYRGHSDTYYNQKFKG (SEQ ID NO:1144); and a VH CDR3 having the amino acid sequence GAIYNGYDVLDN (SEQ ID NO:1145).
In some cases, an anti-BCMA antibody comprises: a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-BCMA antibody comprises: a) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
In some cases, an anti-BCMA antibody (e.g., an antibody referred to in the literature as belantamab) comprises a light chain comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO:1146); and a heavy chain comprising the amino acid sequence:
In some cases, the anti-BCMA antibody has a cancer chemotherapeutic agent linked to the antibody. For example, in some cases, the anti-BCMA antibody is GSK2857916 (belantamab-mafodotin), where monomethyl auristatin F (MMAF) is linked via a maleimidocaproyl linker to the anti-BCMA antibody belantamab.
In some cases, a TTP present in a TMMP is an antibody specific for MUC1. For example, a TTP can be specific for a MUC1 polypeptide present on a cancer cell. In some cases, the TTP is specific for the cleaved form of MUC1; see, e.g., Fessler et al. (2009) Breast Cancer Res. Treat. 118:113. In some cases, the TTP is an antibody specific for a glycosylated MUC1 peptide; see, e.g., Naito et al. (2017) ACS Omega 2:7493; and U.S. Pat. No. 10,017,580.
As one non-limiting example, a TTP can be a single-chain Fv specific for MUC1. See, e.g., Singh et al. (2007) Mol. Cancer Ther. 6:562; Thie et al. (2011) PloSOne 6:e15921; Imai et al. (2004) Leukemia 18:676; Posey et al. (2016) Immunity 44:1444; EP3130607; EP3164418; WO 2002/044217; and US 2018/0112007. In some cases, a TTP is a scFv specific for the MUCI peptide VTSAPDTRPAPGSTAPPAHG (SEQ ID NO:1148). In some cases, a TTP is a scFv specific for the MUCI peptide SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:1149). In some cases, a TTP is a scFv specific for the MUC1 peptide SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:1150). In some cases, a TTP is a scFv specific for the MUCI peptide LAFREGTINVHDVETQFNQY (SEQ ID NO:1151). In some cases, a TTP is a scFv specific for the MUCI peptide SNIKFRPGSVVVQLTLAAFREGTIN (SEQ ID NO:1152).
As an example, an anti-MUCI antibody can comprise: a VH CDR1 having the amino acid sequence RYGMS (SEQ ID NO:1153); a VH CDR2 having the amino acid sequence TISGGGTYIYYPDSVKG (SEQ ID NO:1154); a VH CDR3 having the amino acid sequence DNYGRNYDYGMDY (SEQ ID NO:1155); a VL CDR1 having the amino acid sequence SATSSVSYIH (SEQ ID NO:1156); a VL CDR2 having the amino acid sequence STSNLAS (SEQ ID NO:1157); and a VL CDR3 having the amino acid sequence QQRSSSPFT (SEQ ID NO:1158). See, e.g., US 2018/0112007.
As another example, an anti-MUCI antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO:1159); a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO:1160); a VH CDR3 having the amino acid sequence LGGDNYYEYFDV (SEQ ID NO:1161); a VL CDR1 having the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO:1162); a VL CDR2 having the amino acid sequence LASNLES (SEQ ID NO:1163); and a VL CDR3 having the amino acid sequence QHSRELPFT (SEQ ID NO:1164). See, e.g., US 2018/0112007.
As another example, an anti-MUCI antibody can comprise a VH CDR1 having the amino acid sequence DYAMN (SEQ ID NO:1165); a VH CDR2 having the amino acid sequence VISTFSGNINFNQKFKG (SEQ ID NO:1166); a VH CDR3 having the amino acid sequence SDYYGPYFDY (SEQ ID NO:1167); a VL CDR1 having the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO:1168); a VL CDR2 having the amino acid sequence KVSNRFS (SEQ ID NO:1169); and a VL CDR3 having the amino acid sequence (FQGSHVPFT (SEQ ID NO:1170). See, e.g., US 2018/0112007.
As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO:1159); a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO:1160); a VH CDR3 having the amino acid sequence LGGDNYYEY (SEQ ID NO:1171); a VL CDR1 having the amino acid sequence TASKSVSTSGYSYMH (SEQ ID NO:1172); a VL CDR2 having the amino acid sequence LVSNLES (SEQ ID NO:1173); and a VL CDR3 having the amino acid sequence QHIRELTRSE (SEQ ID NO:1174). See, e.g., US 2018/0112007.
In some cases, a TTP present in a TMMP is an antibody specific for MUC16 (also known as CA125). See, e.g., Yin et al. (2002) Int. J. Cancer 98:737. For example, a TTP can be specific for a MUC16 polypeptide present on a cancer cell. See, e.g., US 2018/0118848; and US 2018/0112008. In some cases, a MUC16-specific TTP is a scFv. In some cases, a MUC16-specific TTP is a nanobody.
As one example, an anti-MUC16 antibody can comprise a VH CDR1 having the amino acid sequence GFTFSNYY (SEQ ID NO:1175); a VH CDR2 having the amino acid sequence ISGRGSTI (SEQ ID NO:1176); a VH CDR3 having the amino acid sequence VKDRGGYSPY (SEQ ID NO:1177); a VL CDR1 having the amino acid sequence QSISTY (SEQ ID NO:1178); a VL CDR2 having the amino acid sequence TAS; and a VL CDR3 having the amino acid sequence QQSYSTPPIT (SEQ ID NO:1179). See, e.g., US 2018/0118848.
In some cases, a TTP present in a TMMP is an antibody specific for claudin-18 isoform 2 (“claudin-18.2”). See, e.g., WO 2013/167259. In some cases, a claudin-18.2-specific TTP is a scFv. In some cases, a claudin-18.2-specific TTP is a nanobody. In some cases, a TPP present in a TMMP is an antibody specific for TEDEVQSYPSKHDYV (SEQ ID NO:1058) or EVQSYPSKHDYV (SEQ ID NO:1059).
As one example, an anti-claudin-18.2 antibody can comprise a VH CDR1 having the amino acid sequence GYTFTDYS (SEQ ID NO:1180); a VH CDR2 having the amino acid sequence INTETGVP (SEQ ID NO:1181); a VH CDR3 having the amino acid sequence ARRTGFDY (SEQ ID NO:1182); a VL CDR1 having the amino acid sequence KNLLHSDGITY (SEQ ID NO:1183); a VL CDR2 having the amino acid sequence RVS; and a VL CDR3 having the amino acid sequence VQVLELPFT (SEQ ID NO:1184).
As another example, an anti-claudin-18.2 antibody can comprise a VH CDR1 having the amino acid sequence GFTFSSYA (SEQ ID NO:1185); a VH CDR2 having the amino acid sequence ISDGGSYS (SEQ ID NO:1186); a VH CDR3 having the amino acid sequence ARDSYYDNSYVRDY (SEQ ID NO:1187); a VL CDR1 having the amino acid sequence QDINTF (SEQ ID NO:1188); a VL CDR2 having the amino acid sequence RTN; and a VL CDR3 having the amino acid sequence LQYDEFPLT (SEQ ID NO:1189).
In embodiments, a TMMP can comprise a heterodimer, wherein: a) the first polypeptide of the heterodimer comprises i) a single-chain polypeptide comprising a peptide epitope (e.g., a viral epitope); a β2M polypeptide; and an MHC class I heavy chain polypeptide, and ii) an antibody heavy chain polypeptide; and wherein b) the second polypeptide of the heterodimer comprises an antibody light chain polypeptide, and wherein either the first polypeptide or the second polypeptide comprises one or more immunomodulatory polypeptides, e.g., on the N-terminus of the antibody light chain polypeptide, on the C-terminus of the antibody light chain polypeptide, or on the C-terminus of the antibody heavy chain polypeptide. A TMMP can thus comprise a heterodimer comprising: a) a first polypeptide comprising: i) a peptide epitope (e.g., a viral epitope); ii) a β2M polypeptide; iii) an MHC class I heavy chain polypeptide; and iv) an antibody heavy chain polypeptide (e.g., an antibody heavy chain comprising, in order from N-terminus to C-terminus: i) a VH polypeptide; ii) an Ig CH1 polypeptide; and iii) an IgG Fc polypeptide); and b) a second polypeptide comprising: i) one or more immunomodulatory polypeptides; and ii) an antibody light chain polypeptide (e.g., an antibody light chain comprising, in order from N-terminus to C-terminus: i) a VL polypeptide; and an Ig kappa light chain constant region polypeptide). A TMMP can comprise a heterodimer comprising: a) a first polypeptide comprising: i) a peptide epitope (e.g., a viral epitope); ii) a β2M polypeptide; iii) an MHC class I heavy chain polypeptide; iv) an antibody heavy chain polypeptide (e.g., an antibody heavy chain comprising, in order from N-terminus to C-terminus: i) a VH polypeptide; ii) an Ig CH1 polypeptide; and iii) an IgG Fc polypeptide); and v) one or more immunomodulatory polypeptides; and b) a second polypeptide comprising an antibody light chain polypeptide (e.g., an antibody light chain comprising, in order from N-terminus to C-terminus: i) a VL polypeptide; and an Ig kappa light chain constant region polypeptide). A TMMP can comprise a heterodimer comprising: a) a first polypeptide comprising: i) a peptide epitope (e.g., a viral epitope); ii) a β2M polypeptide; iii) an MHC class I heavy chain polypeptide; and iv) an antibody heavy chain polypeptide (e.g., an antibody heavy chain comprising, in order from N-terminus to C-terminus: i) a VH polypeptide; ii) an Ig CH1 polypeptide; and iii) an IgG Fc polypeptide); and b) a second polypeptide comprising: i) an antibody light chain polypeptide (e.g., an antibody light chain comprising, in order from N-terminus to C-terminus: i) a VL polypeptide; and an Ig kappa light chain constant region polypeptide); and ii) one or more immunomodulatory polypeptides. Such heterodimers can dimerize (e.g., form homodimers of two of the heterodimers by the formation of disulfide bonds that form between the antibody heavy chain polypeptides of each heterodimer). Such TMMPs are referred to herein as “monoclonal antibody fusions” or “mAb fusions.” The VH polypeptide and the VL polypeptide are on separate polypeptides of the heterodimer; together, the VH polypeptide and the VL polypeptide bind to a cancer epitope and therefore constitute a TTP.
The following are non-limiting examples of mAb fusions.
As one example, a TMMP can comprise: a) a first polypeptide comprising the amino acid sequence depicted in
As another example, a TMMP can comprise: a) a first polypeptide comprising the amino acid sequence depicted in
As another example, a TMMP can comprise: a) a first polypeptide comprising the amino acid sequence depicted in
As noted above, in some cases, a TTP present in a TMMP is a scTCR. A TTP can be a scTCR specific for a peptide/HLA complex on the surface of a cancer cell, where the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen). Amino acid sequences of scTCRs specific for cancer-associated peptides bound to an HLA complex are known in the art. See, e.g., US 2019/0135914; US 2019/0062398; and US 2018/0371049.
A scTCR includes an alpha chain variable region (Vα) and a beta chain variable region (Vβ) covalently linked through a suitable peptide linker sequence. For example, the Vα can be covalently linked to the VP through a suitable peptide linker (L) sequence fused to the C-terminus of the Vα and the N-terminus of the Vβ. An scTCR can have the structure Vα-L-Vβ. An scTCR can have the structure VR-L-Va. An scTCR can also comprise a constant domain (also referred to as constant region). In some cases, an scTCR comprises, in order from N-terminus to C-terminus: i) a TCR α chain variable domain polypeptide; ii) a peptide linker; iii) a TCR β chain variable domain polypeptide; and iv) a TCR β chain constant region extracellular domain polypeptide. In some cases, an scTCR comprises, in order from N-terminus to C-terminus: i) a TCR β chain variable domain polypeptide; ii) a peptide linker; iii) a TCR α chain variable domain polypeptide; and iv) a TCR α chain constant region extracellular domain polypeptide.
Amino acid sequences of scTCRs specific for peptide/HLA complexes, where the peptide is a cancer-associated peptide, are known in the art. See, e.g., US 2019/0135914; US 2019/0062398; US 2018/0371049; US 2019/0144563; and US 2019/0119350.
For example, a scTCR can be specific for an NY-ESO epitope such as an SLLMWITQC (SEQ ID NO:1190) peptide bound to an HLA complex comprising an HLA-A*0201 heavy chain and a β2M polypeptide. As an example, such an scTCR can comprise: i) a TCR α chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence: MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNAS LDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPY (SEQ ID NO:1191), where amino acid 20 can be V or A; amino acid 51 can be Q, P, S, T, or M; amino acid 52 can be S, P, F, or G, amino acid 53 can be S, W, H, or T; amino acid 94 can be P, H, or A; amino acid 95 can be T, L, M, A, Q, Y, E, I, F, V, N, G, S, D, or R; amino acid 96 can be S, L, T, Y, I, Q, V, E, A, W, R, G, H, D, or K; amino acid 97 can be G, D, N, V, S, T, or A; amino acid 98 can be G, P, H, S, T, W, or A; amino acid 99 can be S, T, Y, D, H, V, N, E, G, Q, K, A, I, or R; amino acid 100 can be Y, F, M, or D; amino acid 101 can be I, P, T, or M; and amino acid 103 can be T or A; and ii) a TCR β chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence: MGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPN GYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVL (SEQ ID NO:1192), where amino acid 18 can be M or V; amino acid 50 can be G, V, or I; amino acid 52 can be G or Q; amino acid 53 can be I, T, or M; amino acid 55 can be D or R; amino acid 56 can be Q or R; amino acid 70 can be T or I; amino acid 94 can be Y, N, or F; amino acid 95 can be V or L; and amino acid 97 can be N, G, or D. For example, in some cases, a scTCR can comprise: i) a TCR α chain variable region comprising the amino acid sequence: MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIMSHQREQTSGRLNA SLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPY (SEQ ID NO:1193); and a TCR β chain variable region comprising the amino acid sequence:
As another example, a scTCR can be specific for an HPV epitope (e.g., an HPV peptide of the amino acid sequence YIIFVYIPL (HPV 16 E563-71; SEQ ID NO:1195), KLPQLCTEL (HPV 16 E611-19; SEQ ID NO:1042), TIHEIILECV (HPV 16 E6; SEQ ID NO:1196), YMLDLQPET (HPV 16 E711-19; SEQ ID NO:1049), TLGIVCPI (HPV 16 E786-93) (SEQ ID NO:1055), KCIDFYSRI (HPV 18 E667-75; SEQ ID NO:1197), or FQQLFLNTL (HPV 18 E786-94; SEQ ID NO:1198)) bound to an HLA complex comprising an HL heavy chain and a β2M polypeptide. As an example, such an scTCR can comprise: i) a TCR α chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence: METLLGLLILQLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT SLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRETSGSRLTFGEGTQLTVN PD(SEQ ID NO:1199); and ii) a TCR f chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence:
In some cases, the TTP of a TMMP comprises a contrast agent or a radiolabel, where the contrast agent facilitates imaging of a tumor to which the TMMP binds.
Suitable agents include computed tomography (CT), a positron emission tomography (PET), and single photon emission computed tomography (SPECT) radiotracers. Suitable PET/SPECT contrast agents include, e.g., a positron emitter, for example 11C, 13N, 18F, 82Ru, and 15O. Iodinated CT contrast agents can be used. Suitable contrast agents include gadolinium (Gd), dysprosium, and iron. Gd chelates, such as Gd diethylene triamine pentaacetic acid (GdDTPA), Gd tetraazacyclododecanetetraacetic acid (GdDOTA), polylysine-Gd chelates, and derivatives thereof, can be used. Suitable radioisotopes include 123I (iodine), 18F (fluorine), 99Tc (technetium), 111In (indium), and 67Ga (gallium).
Whether a given peptide (e.g., a peptide that comprises an epitope) binds a class I HLA (comprising an HLA heavy chain and a β2M polypeptide), and, when bound to the HLA complex, can effectively present an epitope to a TCR, can be determined using any of a number of well-known methods. Assays include binding assays and T-cell activation assays. See, e.g., published PCT Application WO 2020/243315 (Cue Biopharma, Inc.).
A TMMP comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a SARS-CoV-2 peptide; and ii) first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide, c) at least one immunomodulatory polypeptide, where the first and/or the second polypeptide comprises the immunomodulatory polypeptide; d) an Ig Fc polypeptide or a non-Ig scaffold, where the first and/or the second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and e) a tumor-targeting polypeptide (TTP), where the first and/or the second polypeptide comprises the TTP. These components can be arranged in any of a variety of configurations. See, e.g.,
Thus, in some cases, a TMMP comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a SARS-CoV-2 peptide; ii) first MHC class I polypeptide; and iii) at least one immunomodulatory polypeptide; b) a second polypeptide comprising a second MHC class I polypeptide; c) an Ig Fc polypeptide or a non-Ig scaffold, where the first and/or the second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and d) a TTP, where the first and/or the second polypeptide comprises the TTP. In other instances, a TMMP comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a SARS-CoV-2 peptide; and ii) first MHC class I polypeptide; b) a second polypeptide comprising: i) a second MHC class I polypeptide; and ii) at least one immunomodulatory polypeptide; c) an Ig Fc polypeptide or a non-Ig scaffold, where the first and/or the second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and d) a TTP, where the first and/or the second polypeptide comprises the TTP. In some cases, a TMMP comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a SARS-CoV-2 peptide; ii) first MHC class I polypeptide; and iii) at least one immunomodulatory polypeptide; b) a second polypeptide comprising: i) a second MHC class I polypeptide; and ii) at least one immunomodulatory polypeptide; c) an Ig Fc polypeptide or a non-Ig scaffold, where the first and/or the second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and d) a TTP, where the first and/or the second polypeptide comprises the TTP. In some cases, the at least one immunomodulatory polypeptide is a wild-type immunomodulatory polypeptide. In other cases, the at least one immunomodulatory polypeptide is a variant immunomodulatory polypeptide that exhibits reduced affinity for a co-immunomodulatory polypeptide, compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the co-immunomodulatory polypeptide. In some cases, a TMMP comprises two immunomodulatory polypeptides, where the two immunomodulatory polypeptides have the same amino acid sequence. In some cases, the TTP is an antibody specific for a cancer-associated antigen, e.g., a cancer-associated antigen present on the surface of a cancer cell. In some cases, the TTP is a scFv or a nanobody. In some cases, the TTP is an antibody specific for a cancer-associated peptide/HLA complex (i.e., an HLA heavy chain and a β2M polypeptide) present on the surface of a cancer cell. In some cases, the TTP is a single-chain T-cell receptor (scTCR) specific for a cancer-associated antigen, e.g., a cancer-associated antigen present on the surface of a cancer cell.
For example, in some cases, a TMMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a SARS-CoV-2 peptide; ii) an optional linker; and iii) a β2M polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a TTP; ii) an optional linker; iii) an MHC class I heavy chain polypeptide; iv) an optional linker; v) an Ig Fc polypeptide; vi) an optional linker; and vii) a MOD. In some cases, a TMMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a SARS-CoV-2 peptide; ii) a linker; and iii) a β2M polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a TTP; ii) a linker; iii) an MHC class I heavy chain polypeptide; iv) a linker; v) an Ig Fc polypeptide; vi) a linker; and vii) a MOD. In some cases, a TMMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a SARS-CoV-2 peptide; ii) a linker; and iii) a β2M polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a TTP; ii) a linker; iii) an MHC class I heavy chain polypeptide; iv) a linker; v) an Ig Fc polypeptide; vi) a linker; vii) a first MOD; viii) a linker; and ix) a second MOD. In some cases, the HLA heavy chain comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*0201, an HLA-A*1101, an HLA-A*2402, or an HLA-A*3303 amino acid sequence depicted in any one of
In some cases, a TMMP comprises an HLA-A*0201 polypeptide having an amino acid sequence having at least at least 70%, 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 amino acid sequence of the HLA-A*0201 polypeptide depicted in
In some cases, a TMMP comprises an HLA-A*1101 polypeptide having an amino acid sequence having at least at least 70%, 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 amino acid sequence of the HLA-A*1101 polypeptide depicted in
In some cases, a TMMP comprises an HLA-A*2402 polypeptide having an amino acid sequence having at least at least 70%, 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 amino acid sequence of the HLA-A*2402 polypeptide depicted in
In some cases, a TMMP comprises an HLA-E*0101 polypeptide having an amino acid sequence having at least at least 70%, 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 amino acid sequence of the HLA-E*0101 polypeptide depicted in
In some cases, a TMMP comprises an HLA-E*0103 polypeptide having an amino acid sequence having at least at least 70%, 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 amino acid sequence of the HLA-E*0103 polypeptide depicted in
As one non-limiting example, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising the amino acid sequence depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising the amino acid sequence depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising the amino acid sequence depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising the amino acid sequence depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising the amino acid sequence depicted in
As one non-limiting example, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in
As one non-limiting example, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in
As one non-limiting example, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in
As one non-limiting example, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in
As one non-limiting example, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in
As one non-limiting example, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in
As noted above, in some cases, a TMMP comprises an HLA-E heavy chain polypeptide. In some cases, the TMMP comprises a SARS-CoV-2 peptide having the amino acid sequence YLQPRTLFL (SEQ ID NO:887). In some cases, the TMMP comprises a SARS-CoV-2 peptide having the amino acid sequence YLQPRTLIL (SEQ ID NO:1201). In some cases, the TMMP comprises a SARS-CoV-2 peptide having the amino acid sequence YLQPRTLVL (SEQ ID NO:1202). As one non-limiting example, a TMMP comprises: a) a first polypeptide comprising the amino acid sequence depicted in
As one non-limiting example, a TMMP comprises: a) a first polypeptide comprising: i) a SARS-CoV2 peptide that has the amino acid sequence YLQPRTFLL (SEQ ID NO:218); ii) a linker comprising the amino acid sequence GCGGS(GGGS)2 (SEQ ID NO:1203); and iii) a β2M polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following β2M amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLL YYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:2), where amino acid 12 is a Cys; and b) a second polypeptide comprising: i) an anti-CD19 scFv comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following anti-CD19 scFv amino acid sequence: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRF SGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQ LQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFK GKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO:872); ii) a linker, e.g., where the linker has the sequence GGGGS (SEQ ID NO:873); iii) an HLA-A*0201 polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following HLA-A*0201 amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGET RKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIA LKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPK THMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVV VPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:18), where amino acid 84 is a Cys and where amino acid 236 is a Cys; iv) a linker, e.g., where the linker has the sequence AAAGG (SEQ ID NO:874); v) an IgG Fc polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following IgG Fc amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:877), where amino acid 14 is an Ala and where amino acid 15 is an Ala; vi) a linker, e.g., where the linker has the amino acid sequence (GGGGS)3 (SEQ ID NO:875); vii) a first MOD, where the first MOD comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following variant IL-2 amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELK PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIST LT (SEQ ID NO:878), e.g., where amino acid 16 is an Ala and where amino acid 42 is an Ala; viii) a linker, e.g., where the linker has the amino acid sequence (GGGGS)4 (SEQ ID NO:876); and ix) a second MOD, where the second MOD comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following variant IL-2 amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELK PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIST LT (SEQ ID NO:878), e.g., where amino acid 16 is an Ala and where amino acid 42 is an Ala. As an example, in some cases, a TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of the “4700” construct depicted in
In some cases, a TMMP comprises: a)) a first polypeptide comprising: i) a peptide epitope having a length of from 4 amino acids to 25 amino acids (e.g., from 6 to 12 amino acids; from 8 to 12 amino acids; etc.); ii) a linker having the amino acid sequence GCGGS(GGGGS)2 (SEQ ID NO:882); and iii) a β2M polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the β2M amino acid sequence depicted in
In some cases, a TMMP comprises: a) a first polypeptide comprising: i) a peptide epitope having a length of from 4 amino acids to 25 amino acids (e.g., from 6 to 12 amino acids; from 8 to 12 amino acids; etc.); ii) a linker having the amino acid sequence GCGGS(GGGGS)2 (SEQ ID NO:882); and iii) a β2M polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the β2M amino acid sequence depicted in
Although embodiments including a SARS-CoV-2 are illustrated herein, as discussed above, peptide epitopes from other pathogens could be employed in TMMPs that also comprise one or more MODs. For example, other suitable epitopes include, but are not limited to, epitopes present in an infectious disease agent, e.g., an epitope presented by a virus-encoded polypeptide or bacteria. Examples of viral infectious disease agents include, e.g., Adenoviruses, Adeno-associated virus, Alphaviruses (Togaviruses), Eastern equine encephalitis virus, Eastern equine encephalomyelitis virus, Venezuelan equine encephalomyelitis vaccine strain TC-83, Western equine encephalomyelitis virus, Arenaviruses, Lymphocytic choriomeningitis virus (non-neurotropic strains), Tacaribe virus complex, Bunyaviruses, Bunyamwera virus, Rift Valley fever virus vaccine strain MP-12, Chikungunya virus, Calciviruses, Coronaviruses, Cowpox virus, Flaviviruses (Togaviruses)—Group B Arboviruses, Dengue virus serotypes 1, 2, 3, and 4, Yellow fever virus vaccine strain 17D, Hepatitis A, B, C, D, and E viruses, the Cytomegalovirus, Epstein Barr virus, Eastern Equine encephalitis virus, Herpes simplex types 1 and 2, Herpes zoster, Human herpesvirus types 6 and 7, hepatitis C virus (HVC), hepatitis B virus (HBV), Influenza viruses types A, B, and C, Papovaviruses, Newcastle disease virus, Measles virus, Mumps virus, Parainfluenza viruses types 1, 2, 3, and 4, polyomaviruses (JC virus, BK virus), Respiratory syncytial virus, Human parvovirus (B 19), Coxsackie viruses types A and B, Echoviruses, Polioviruses, Rhinoviruses, Alastrim (Variola minor virus), Smallpox (Variola major virus), Whitepox Reoviruses, Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus), Rabies virus, Vesicular stomatitis virus, Rubivirus (rubella), Semliki Forest virus, St. Louis encephalitis virus, Venezuelan equine encephalitis virus, Venezuelan equine encephalomyelitis virus, Arenaviruses (a.k.a. South American Hemorrhagic Fever virus), Flexal, Lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantaviruses including Hantaan virus, Rift Valley fever virus, Japanese encephalitis virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus (HIV) types 1 and 2, Human T cell lymphotropic virus (HTLV) types 1 and 2, Simian immunodeficiency virus (SIV), Vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia, Crimean-Congo hemorrhagic fever virus, Ebola viruses, Marburg virus, Tick-borne encephalitis virus complex (flavi) including Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian Spring Summer encephalitis viruses, Herpesvirus simiae (Herpes B or Monkey B virus), Cercopithecine herpesvirus 1 (Herpes B virus), Equine morbillivirus (Hendra and Hendra-like viruses), Nipah virus, Variola major virus (Smallpox virus), Variola minor virus (Alastrim), African swine fever virus, African horse sickness virus, Akabane virus, Avian influenza virus (highly pathogenic), Blue tongue virus, Camel pox virus, Classical swine fever virus, Cowdria ruminantium (heartwater), Foot and mouth disease virus, Goat pox virus, Japanese encephalitis virus, Lumpy skin disease virus, Malignant catarrhal fever virus, Menangle virus, Newcastle disease virus (VVND), Vesicular stomatitis virus (exotic), and Zika virus. Antigens encoded by such viruses are known in the art; a peptide epitope suitable for use in a TMMP of the present disclosure can include a peptide from any known viral antigen. In some cases, a viral epitope is an epitope present in a viral antigen encoded by a virus other than SARS-CoV-2 that infects a majority of the human population, where such viruses include, e.g., cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papilloma virus, adenovirus, and the like. See, e.g., published PCT application WO2020/243315 (Cue Biopharma, Inc.) at paragraphs [00321]-[00338]. Accordingly, this disclosure expressly contemplates TMMPs comprising an epitope other than a SARS-CoV-2 epitope.
Tmmps without an Immunomodulatory Polypeptide
The present disclosure provides a TMMP comprising: a) a first polypeptide; and b) a second polypeptide, wherein the TMMP comprises: a peptide epitope; a first MHC polypeptide; a second MHC polypeptide; an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold; and a TTP, where the TMMP does not include an immunomodulatory polypeptide (e.g., does not comprise a MOD as set forth in Table 2; e.g., does not comprise an IL-2 polypeptide, a 4-1BBL polypeptide, etc.). Such a TMMP may also be referred to herein as a “MODless TMMP.” The components of a MODless TMMP (e.g., peptide epitope, first MHC polypeptide, second MHC polypeptide, Ig Fc polypeptide or non-Ig scaffold, and TTP) can be arranged in a variety of configurations. The components (other than MODs) are as described above.
In some cases, the present disclosure provides a protein comprising at least one heterodimer comprising: a) a first polypeptide comprising: i) a SARS-CoV-2 peptide, wherein the SARS-CoV-2 peptide has a length of from about 4 amino acids to about 25 amino acids, e.g., from 8 to 12 amino acids; and a β2M polypeptide; b) a second polypeptide an MHC class I heavy chain polypeptide; c) an Ig Fc polypeptide or a non-Ig scaffold, where the first or the second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and d) a TTP, where the first and/or the second polypeptide comprises the TTP, wherein the protein does not include an immunomodulatory polypeptide. One or more of the individual components of the first and/or second polypeptide can be linked together by one or more linkers. Suitable SARS-CoV-2 peptides, MHC class I heavy chain polypeptides, and Ig Fc polypeptides are described above. Such a protein can also be referred to as a “MOD-less TMMP.”
In some cases, a MOD-less TMMP is dimerized. Thus, present disclosure thus provides a protein that is a dimerized MOD-less TMMP comprising two MOD-less TMMPs that are covalently linked to each other. The covalent linkage of the dimer can be one or more disulfide bonds between an Ig Fc polypeptide in the first MOD-less TMMP and an Ig Fc polypeptide in the second MOD-less TMMP.
In some cases, a MODless TMMP comprises: a) a first polypeptide comprising: (i) a peptide epitope, where the peptide epitope is a SARS-CoV-2 peptide; and (ii) a first MHC polypeptide, where the first MHC polypeptide is a β2M polypeptide; and b) a second polypeptide comprising: (i) a TTP; (ii) a second MHC polypeptide, where the second MHC polypeptide is an MHC class I heavy chain polypeptide; and (iii) an Ig Fc polypeptide. A peptide linker may be interposed between one or more of: (i) the peptide epitope and the β2M polypeptide; (ii) the MHC class I heavy chain polypeptide and the TTP; (iii) the MHC class I heavy chain polypeptide and the Ig Fc polypeptide. In some cases, the first polypeptide comprises, in order from N-terminus to C-terminus: (i) a SARS-CoV-2 peptide; and (ii) a β2M polypeptide. In some cases, the first polypeptide comprises, in order from N-terminus to C-terminus: (i) a SARS-CoV-2 peptide; (ii) a peptide linker, where the peptide linker optionally may include a Cys residue; and (iii) a β2M polypeptide. In some cases, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) a TTP; (ii) an optional linker; (iii) an MHC class I heavy chain polypeptide; (iv) an optional linker; and (v) an Ig Fc polypeptide. In some cases, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) an MHC class I heavy chain polypeptide; (ii) an optional linker; (iii) an Ig Fc polypeptide; (iv) an optional linker; and (v) a TTP. In some cases, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) an MHC class I heavy chain polypeptide; (ii) an optional linker; (iii) a TTP; (iv) an optional linker; and (v) an Ig Fc polypeptide. In some cases, the β2M polypeptide comprises a Cys residue, e.g., a Cys residue at position 12 according to the amino acid numbering of the β2M amino acid sequence depicted in
The following are non-limiting examples of MODless TMMPs.
In some cases, a MODless TMMP comprises: a) a first polypeptide comprising: (i) a SARS-CoV-2 peptide; and (ii) a β2M polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following β2M amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLL YYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:2), where amino acid 12 is a Cys; and b) a second polypeptide comprising: (i) an anti-CD19 scFv polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the anti-CD19 scFv polypeptide amino acid sequence depicted in
In some cases, a MODless TMMP comprises: a) a first polypeptide comprising: (i) a SARS-CoV-2 peptide; and (ii) a β2M polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following β2M amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLL YYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:2), where amino acid 12 is a Cys; and b) a second polypeptide comprising: (i) an anti-CD19 scFv polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the anti-CD19 scFv polypeptide amino acid sequence depicted in
Although MODless embodiments including a SARS-CoV-2 are illustrated herein, those skilled in the art will readily understand that other peptide epitopes could be employed in MODless TMMPs. For example, other suitable epitopes include, but are not limited to, epitopes present in an infectious disease agent, e.g., an epitope presented by a virus-encoded polypeptide. Examples of viral infectious disease agents include, e.g., Adenoviruses, Adeno-associated virus, Alphaviruses (Togaviruses), Eastern equine encephalitis virus, Eastern equine encephalomyelitis virus, Venezuelan equine encephalomyelitis vaccine strain TC-83, Western equine encephalomyelitis virus, Arenaviruses, Lymphocytic choriomeningitis virus (non-neurotropic strains), Tacaribe virus complex, Bunyaviruses, Bunyamwera virus, Rift Valley fever virus vaccine strain MP-12, Chikungunya virus, Calciviruses, Coronaviruses, Cowpox virus, Flaviviruses (Togaviruses)—Group B Arboviruses, Dengue virus serotypes 1, 2, 3, and 4, Yellow fever virus vaccine strain 17D, Hepatitis A, B, C, D, and E viruses, the Cytomegalovirus, Epstein Barr virus, Eastern Equine encephalitis virus, Herpes simplex types 1 and 2, Herpes zoster, Human herpesvirus types 6 and 7, hepatitis C virus (HVC), hepatitis B virus (HBV), Influenza viruses types A, B, and C, Papovaviruses, Newcastle disease virus, Measles virus, Mumps virus, Parainfluenza viruses types 1, 2, 3, and 4, polyomaviruses (JC virus, BK virus), Respiratory syncytial virus, Human parvovirus (B 19), Coxsackie viruses types A and B, Echoviruses, Polioviruses, Rhinoviruses, Alastrim (Variola minor virus), Smallpox (Variola major virus), Whitepox Reoviruses, Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus), Rabies virus, Vesicular stomatitis virus, Rubivirus (rubella), Semliki Forest virus, St. Louis encephalitis virus, Venezuelan equine encephalitis virus, Venezuelan equine encephalomyelitis virus, Arenaviruses (a.k.a. South American Hemorrhagic Fever virus), Flexal, Lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantaviruses including Hantaan virus, Rift Valley fever virus, Japanese encephalitis virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus (HIV) types 1 and 2, Human T cell lymphotropic virus (HTLV) types 1 and 2, Simian immunodeficiency virus (SIV), Vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia, Crimean-Congo hemorrhagic fever virus, Ebola viruses, Marburg virus, Tick-borne encephalitis virus complex (flavi) including Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian Spring Summer encephalitis viruses, Herpesvirus simiae (Herpes B or Monkey B virus), Cercopithecine herpesvirus 1 (Herpes B virus), Equine morbillivirus (Hendra and Hendra-like viruses), Nipah virus, Variola major virus (Smallpox virus), Variola minor virus (Alastrim), African swine fever virus, African horse sickness virus, Akabane virus, Avian influenza virus (highly pathogenic), Blue tongue virus, Camel pox virus, Classical swine fever virus, Cowdria ruminantium (heartwater), Foot and mouth disease virus, Goat pox virus, Japanese encephalitis virus, Lumpy skin disease virus, Malignant catarrhal fever virus, Menangle virus, Newcastle disease virus (VVND), Vesicular stomatitis virus (exotic), and Zika virus. Antigens encoded by such viruses are known in the art; a peptide epitope suitable for use in a TMMP of the present disclosure can include a peptide from any known viral antigen. In some cases, a viral epitope is an epitope present in a viral antigen encoded by a virus that infects a majority of the human population, where such viruses include, e.g., cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papilloma virus, adenovirus, and the like. See, e.g., published PCT application WO2020/243315 (Cue Biopharma, Inc.) at paragraphs [00321]-[00338]. Accordingly, this disclosure expressly contemplates MODless TMMPs comprising an epitope other than a SARS-CoV-2 epitope.
For example, in some cases, a MODless TMMP comprises: a) a first polypeptide comprising: (i) a viral peptide epitope, e.g., the peptide epitope is a CMV peptide; and (ii) a first MHC polypeptide, where the first MHC polypeptide is a β2M polypeptide; and b) a second polypeptide comprising: (i) a TTP; (ii) a second MHC polypeptide, where the second MHC polypeptide is an MHC class I heavy chain polypeptide; and (iii) an Ig Fc polypeptide. A peptide linker may be interposed between one or more of: (i) the peptide epitope and the β2M polypeptide; (ii) the MHC class I heavy chain polypeptide and the TTP; (iii) the MHC class I heavy chain polypeptide and the Ig Fc polypeptide. In some cases, the first polypeptide comprises, in order from N-terminus to C-terminus: (i) a CMV peptide; and (ii) a β2M polypeptide. In some cases, the first polypeptide comprises, in order from N-terminus to C-terminus: (i) a viral peptide epitope, e.g., a CMV peptide epitops; (ii) a peptide linker, where the peptide linker may include a Cys residue; and (iii) a β2M polypeptide. In some cases, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) a TTP; (ii) an optional linker; (iii) an MHC class I heavy chain polypeptide; (iv) an optional linker; and (v) an Ig Fc polypeptide. In some cases, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) an MHC class I heavy chain polypeptide; (ii) an optional linker; (iii) an Ig Fc polypeptide; (iv) an optional linker; and (v) a TTP. In some cases, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) an MHC class I heavy chain polypeptide; (ii) an optional linker; (iii) a TTP; (iv) an optional linker; and (v) an Ig Fc polypeptide. In some cases, the β2M polypeptide comprises a Cys residue, e.g., a Cys residue at position 12 according to the amino acid numbering of the β2M amino acid sequence depicted in
In some cases, a MODless TMMP comprises: a) a first polypeptide comprising: (i) a CMV peptide; and (ii) a β2M polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following β2M amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLL YYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:2), where amino acid 12 is a Cys;
The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a TMMP. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a TMMP.
The present disclosure provides nucleic acids comprising nucleotide sequences encoding a TMMP. In some cases, the individual polypeptide chains of a TMMP are encoded in separate nucleic acids. In some cases, all polypeptide chains of a TMMP are encoded in a single nucleic acid. In some cases, a first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a TMMP; and a second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of a TMMP. In some cases, single nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a TMMP and a second polypeptide of a TMMP.
The present disclosure provides nucleic acids comprising nucleotide sequences encoding a TMMP. As noted above, in some cases, the individual polypeptide chains of a TMMP are encoded in separate nucleic acids. In some cases, nucleotide sequences encoding the separate polypeptide chains of a TMMP are operably linked to transcriptional control elements, e.g., promoters, such as promoters that are functional in a eukaryotic cell, where the promoter can be a constitutive promoter or an inducible promoter.
For example, the present disclosure provides a first nucleic acid and a second nucleic acid, where the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a TMMP, where the first polypeptide comprises, in order from N-terminus to C-terminus: a) a peptide epitope; b) a first MHC polypeptide; and c) an immunomodulatory polypeptide (e.g., a reduced-affinity variant, as described above); and where the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of a TMMP, where the second polypeptide comprises, in order from N-terminus to C-terminus: a) a second MHC polypeptide; b) an Ig Fc polypeptide; and c) a TTP. Suitable peptide epitopes, MHC polypeptides, immunomodulatory polypeptides, Ig Fc polypeptides, and TTPs are described above. In some cases, the nucleotide sequences encoding the first and the second polypeptides are operably linked to transcriptional control elements. In some cases, the transcriptional control element is a promoter that is functional in a eukaryotic cell. In some cases, the nucleic acids are present in separate expression vectors.
The present disclosure provides a first nucleic acid and a second nucleic acid, where the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a TMMP, where the first polypeptide comprises, in order from N-terminus to C-terminus: a) a peptide epitope; and b) a first MHC polypeptide; and where the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of a TMMP, where the second polypeptide comprises, in order from N-terminus to C-terminus: a) an immunomodulatory polypeptide (e.g., a reduced-affinity variant as described above); b) a second MHC polypeptide; c) an Ig Fc polypeptide; and d) a TTP. Suitable peptide epitopes, MHC polypeptides, immunomodulatory polypeptides, and Ig Fc polypeptides, are described above. In some cases, the nucleotide sequences encoding the first and the second polypeptides are operably linked to transcriptional control elements. In some cases, the transcriptional control element is a promoter that is functional in a eukaryotic cell. In some cases, the nucleic acids are present in separate expression vectors.
The present disclosure provides a nucleic acid comprising nucleotide sequences encoding at least the first polypeptide and the second polypeptide of a TMMP. In some cases, where a TMMP includes a first, second, and third polypeptide, the nucleic acid includes a nucleotide sequence encoding the first, second, and third polypeptides. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of a TMMP includes a proteolytically cleavable linker interposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of a TMMP includes an internal ribosome entry site (IRES) interposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of a TMMP includes a ribosome skipping signal (or cis-acting hydrolase element, CHYSEL) interposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. Examples of nucleic acids are described below, where a proteolytically cleavable linker is provided between nucleotide sequences encoding the first polypeptide and the second polypeptide of a TMMP; in any of these embodiments, an IRES or a ribosome skipping signal can be used in place of the nucleotide sequence encoding the proteolytically cleavable linker.
In some cases, a first nucleic acid (e.g., a recombinant expression vector, an mRNA, a viral RNA, etc.) comprises a nucleotide sequence encoding a first polypeptide chain of a TMMP; and a second nucleic acid (e.g., a recombinant expression vector, an mRNA, a viral RNA, etc.) comprises a nucleotide sequence encoding a second polypeptide chain of a TMMP. In some cases, the nucleotide sequence encoding the first polypeptide, and the second nucleotide sequence encoding the second polypeptide, are each operably linked to transcriptional control elements, e.g., promoters, such as promoters that are functional in a eukaryotic cell, where the promoter can be a constitutive promoter or an inducible promoter.
The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide, where the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) a peptide epitope; b) a first MHC polypeptide; c) an immunomodulatory polypeptide (e.g., a reduced-affinity variant as described above); d) a proteolytically cleavable linker; e) a second MHC polypeptide; f) an Ig Fc polypeptide; and g) a TTP. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide, where the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) a first leader peptide; b) the epitope; c) the first MHC polypeptide; d) the immunomodulatory polypeptide (e.g., a reduced-affinity variant as described above); e) the proteolytically cleavable linker; f) a second leader peptide; g) the second MHC polypeptide; h) the Ig Fc polypeptide; and i) the TTP. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide, where the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) an epitope; b) a first MHC polypeptide; c) a proteolytically cleavable linker; d) an immunomodulatory polypeptide (e.g., a reduced-affinity variant as described above); e) a second MHC polypeptide; f) an Ig Fc polypeptide; and g) a TTP.
The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide, where the recombinant polypeptide comprises, in order from N-terminus to C-terminus: a) an epitope; b) a first MHC polypeptide; c) a proteolytically cleavable linker; d) an immunomodulatory polypeptide (e.g., a reduced-affinity variant as described above); e) a TTP; f) a second MHC polypeptide; and g) an Ig Fc polypeptide.
In some cases, the first leader peptide and the second leader peptide are a 32-M leader peptide. In some cases, the nucleotide sequence is operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that is functional in a eukaryotic cell.
Suitable MHC polypeptides are described above. In some cases, the first MHC polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide. In some cases, the β2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to a β2M amino acid sequence depicted in
Suitable Fc polypeptides are described above. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide. In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to an amino acid sequence depicted in
Suitable immunomodulatory polypeptides are described above.
Suitable proteolytically cleavable linkers are described above. In some cases, the proteolytically cleavable linker comprises an amino acid sequence selected from: a) LEVLFQGP (SEQ ID NO:1204); b) ENLYTQS (SEQ ID NO:1205); c) DDDDK (SEQ ID NO:1206); d) LVPR (SEQ ID NO:1207); and e) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:1208).
In some cases, a linker between the epitope and the first MHC polypeptide comprises a first Cys residue, and the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, such that the first and the second Cys residues provide for a disulfide linkage between the linker and the second MHC polypeptide. In some cases, first MHC polypeptide comprises an amino acid substitution to provide a first Cys residue, and the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, such that the first Cys residue and the second Cys residue provide for a disulfide linkage between the first MHC polypeptide and the second MHC polypeptide.
The present disclosure provides recombinant expression vectors comprising nucleic acids.
Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available. See, e.g., published PCT Application WO 2020/243315 (Cue Biopharma, Inc.).
The present disclosure provides a genetically modified host cell, where the host cell is genetically modified with a nucleic acid.
Suitable host cells include eukaryotic cells, such as yeast cells, insect cells, and mammalian cells. In some cases, the host cell is a cell of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.
In some cases, the host cell is a mammalian cell that has been genetically modified such that it does not synthesize endogenous MHC β2-M.
In some cases, the host cell is a mammalian cell that has been genetically modified such that it does not synthesize endogenous MHC Class I heavy chain. In some cases, the host cell is a mammalian cell that has been genetically modified such that it does not synthesize endogenous MHC β2-M and such that it does not synthesize endogenous MHC Class I heavy chain.
The present disclosure provides methods of producing a TMMP. The methods generally involve culturing, in a culture medium, a host cell that is genetically modified with one or more nucleic acids (e.g., one or more recombinant expression vectors) comprising nucleotide sequences encoding the TMMP; and isolating the TMMP from the genetically modified host cell and/or the culture medium. A host cell that is genetically modified with one or more nucleic acids (e.g., one or more recombinant expression vectors) comprising nucleotide sequences encoding the TMMP is also referred to as an “expression host.” As noted above, in some cases, the individual polypeptide chains of a TMMP are encoded in separate nucleic acids (e.g., separate recombinant expression vectors). In some cases, all polypeptide chains of a TMMP are encoded in a single nucleic acid (e.g., a single recombinant expression vector).
Isolation of the TMMP from the expression host cell (e.g., from a lysate of the expression host cell) and/or the culture medium in which the host cell is cultured, can be carried out using standard methods of protein purification.
For example, a lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. Alternatively, where the TMMP is secreted from the expression host cell into the culture medium, the TMMP can be purified from the culture medium using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some cases, the compositions which are used will comprise at least 80% by weight of the desired product (TMMP), at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. The percentages can be based upon total protein.
In some cases, e.g., where the TMMP comprises an affinity tag, the TMMP can be purified using an immobilized binding partner of the affinity tag.
The present disclosure provides compositions, including pharmaceutical compositions, comprising a TMMP of the present disclosure. The present disclosure provides compositions, including pharmaceutical compositions, comprising a TMMP.
A wide variety of pharmaceutically acceptable ingredients are known in the art and hence are not discussed in detail herein. Moreover, pharmaceutically acceptable ingredients and compositions have been amply described in a variety of publications, including, but not limited to, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. Many other publications describing preparation of biopharmaceutical compositions may be consulted.
The composition may be formulated according to the various routes of administration described below. Generally speaking, TMMPs of this disclosure, e.g., a homodimer comprising two TMMPs, will be an aqueous liquid and typically will be administered via an intravenous infusion. In some cases, the pharmaceutical composition comprising the TMMP can be admixed with saline (e.g., 0.9% NaCl) prior to IV administration. Thus, the present disclosure provides a sterile composition comprising: a) a TMMP of the present disclosure; and b) saline (e.g., 0.9% NaCl). Alternatively, it may be administered neat via an intravenous infusion, i.e., without further dilution. Alternatively, the pharmaceutical composition may be formulated so as to be administered by injection.
Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. As noted above, a pharmaceutical composition comprising a TMMP typically will be administered intravenously, but may also be administered by other routes that involve injection.
The present disclosure provides compositions, e.g., pharmaceutical compositions, comprising a nucleic acid or a recombinant expression vector. A wide variety of pharmaceutically acceptable excipients is known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.
The present disclosure provides a method of selectively modulating the activity of an epitope-specific T cell (e.g., a T cell comprising a TCR specific for SARS-CoV-2 (e.g., a SARS-CoV-2 peptide in a complex with MHC polypeptides); and/or a T-cell specific for a cancer-associated antigen), the method comprising contacting the T cell with a TMMP, where contacting the T cell with a TMMP selectively modulates the activity of the epitope-specific T cell. In some cases, the contacting occurs in vitro. In some cases, the contacting occurs in vivo. In some cases, the contacting occurs ex vivo.
Where a TMMP includes an immunomodulatory polypeptide that is an activating polypeptide, contacting the T cell with the TMMP activates the epitope-specific T cell.
In some instances, a TMMP includes: i) an immunomodulatory polypeptide that is an activating polypeptide; ii) a SARS-CoV-2 peptide; and iii) a TTP that targets a cancer cell; and the TMMP is contacted with a T cell that is specific for the SARS-CoV-2 peptide present in the TMMP. In these instances, contacting the SARS-CoV-2-specific T cell with the TMMP activates the SARS-CoV-2-specific T cell and/or increases proliferation of the SARS-CoV-2-specific T cell. In some cases, contacting the SARS-CoV-2-specific T cell with the TMMP increases the number and/or cytotoxic activity of the T cell toward a cancer cell that is targeted by the TTP present in the TMMP. As one non-limiting example, where a TMMP comprises: i) an immunomodulatory polypeptide that is an activating polypeptide (e.g., an IL-2 polypeptide); ii) a SARS-CoV-2 peptide as the peptide epitope; and iii) a TTP that is a scFv that binds Her2, contacting the TMMP with a cytotoxic T cell that binds the SARS-CoV-2 peptide activates the T cell and increases its cytotoxic activity toward a Her2-expressing cancer cell.
The present disclosure provides a method of modulating an immune response in an individual, the method comprising administering to the individual an effective amount of a TMMP. Administering the TMMP induces an epitope-specific T cell response (e.g., a SARS-CoV-2-specific response) and an epitope-non-specific T cell response, where the ratio of the epitope-specific T cell response to the epitope-non-specific T cell response is at least 2:1 or greater, e.g. at least 5:1, 10:1, at least 25:1 or greater. In some cases, the modulating increases a cytotoxic T-cell response to a cancer cell, e.g., a cancer cell expressing an antigen that is targeted by the TTP present in the TMMP.
“Modulating the activity” of a T cell can include, for example, one or more of: i) activating a cytotoxic (e.g., CD8+) T cell; ii) inducing cytotoxic activity of a cytotoxic (e.g., CD8+) T cell; and/or iii) inducing production and release of a cytotoxin (e.g., a perforin; a granzyme; a granulysin) by a cytotoxic (e.g., CD8+) T cell.
The present disclosure provides a method of delivering a costimulatory (i.e., immunomodulatory) polypeptide selectively to target T cell, the method comprising contacting a mixed population of T cells with a TMMP, where the mixed population of T cells comprises the target T cell and non-target T cells, where the target T cell is specific for the epitope present within the TMMP (e.g., where the target T cell is specific for the epitope present within the TMMP), and where the contacting step delivers the one or more costimulatory polypeptides (immunomodulatory polypeptides) present within the TMMP to the target T cell. In some cases, the population of T cells is in vitro. In some cases, the population of T cells is in vivo in an individual. In some cases, the method comprises administering the TMMP to the individual. In some case, the T cell is a cytotoxic T cell. In some cases, the mixed population of T cells is an in vitro population of mixed T cells obtained from an individual, and the contacting step results in activation and/or proliferation of the target T cell, generating a population of activated and/or proliferated target T cells; in some of these instances, the method further comprises administering the population of activated and/or proliferated target T cells to the individual.
The present disclosure provides a method of detecting, in a mixed population of T cells obtained from an individual, the presence of a target T cell that binds an epitope of interest (e.g., a SARS-CoV-2 epitope), the method comprising: a) contacting in vitro the mixed population of T cells with a TMMP, wherein the TMMP comprises the epitope of interest (e.g., the SARS-CoV-2 epitope); and b) detecting activation and/or proliferation of T cells in response to said contacting, wherein activated and/or proliferated T cells indicates the presence of the target T cell.
The present disclosure provides a method of treatment of an individual, the method comprising administering to the individual an amount of a TMMP (e.g., a homodimer TMMP), or one or more nucleic acids encoding the TMMP, effective to treat the individual. Also provided is a TMMP for use in a method of treatment of the human or animal body. In some cases, a treatment method comprises administering to an individual in need thereof one or more recombinant expression vectors comprising nucleotide sequences encoding a TMMP. In some cases, a treatment method comprises administering to an individual in need thereof one or more mRNA molecules comprising nucleotide sequences encoding a TMMP. In some cases, a treatment method comprises administering to an individual in need thereof a TMMP. Conditions that can be treated include, e.g., cancer and autoimmune disorders, as described below.
A TMMP can both: 1) modulate the activity of an epitope-specific T cell (e.g., a T cell specific for the epitope present in the TMMP); and 2) target the TMMP to a target cell. For example, the TTP can cause the TMMP to bind to a cancer cell, where the TMMP engages with and activates a T cell specific for the epitope, e.g., the cytotoxic activity of the T cell is increased, which in turn leads to killing of the cancer cell. For example, in some cases, a TMMP:1) targets the TMMP to a cancer cell, and 2) induces a cytotoxic T cell response to a viral epitope (e.g., SARS-CoV-2 peptide) present in the TMMP.
As noted above, depending on the affinity for a wild-type MOD for its co-MOD, the combination of the reduced affinity of the MOD for its co-MOD, and the affinity of the epitope for a TCR, provides for enhanced selectivity of a TMMP. Thus, for example, a TMMP 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 co-immunomodulatory 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. Variant MODs may be advantageous in multiple circumstance, e.g., where a wild-type MOD has a relatively high affinity for a co-MOD (e.g., IL-2 for IL-2R) and/or can have multiple effects upon binding (e.g., IL-2, which can both activate epitope-specific T cells and upregulate the production of Tregs), or where a MOD can bind to multiple co-MODs (e.g., CD80, which can bind both CD28 and CTLA4). In such cases, reducing the affinity for a co-MOD may be advantageous by decreasing off-target binding of the TMMP, increasing the desired activation of the TMMP and/or decreasing or substantially eliminating the undesired activation of the TMMP.
The present disclosure provides a method of selectively modulating the activity of an epitope-specific T cell in an individual, the method comprising administering to the individual an effective amount of a TMMP, or one or more nucleic acids (e.g., expression vectors; mRNA; etc.) comprising nucleotide sequences encoding the TMMP, where the TMMP selectively modulates the activity of the epitope-specific T cell in the individual. Selectively modulating the activity of an epitope-specific T cell can treat a disease or disorder in the individual. Thus, the present disclosure provides a treatment method comprising administering to an individual in need thereof an effective amount of a TMMP.
Cancers that can be treated with a method include any cancer that can be targeted with a TTP. Cancers that can be treated with a method include carcinomas, sarcomas, melanoma, leukemias, lymphomas and multiple myeloma. Cancers that can be treated with a method include solid tumors, and cancers that begin in blood-forming tissue, i.e., hematological cancers such as leukemias, lymphomas and multiple myeloma. Cancers that can be treated with a method include metastatic cancers.
Cancers that that can be treated with a method include cancers that have escaped/evaded the immune system through HLA loss. Such cancers, which lack cell-surface expression of the class I major histocompatibility complex, thus can escape recognition by cancer-specific cytotoxic T cells. Because the anti-cancer effect of the TMMPs described herein does not depend on recognition of the cancer cells by cancer-specific cytotoxic T cells, but rather on the recognition by T cells that have a TCR that is specific for the pMHC on the TMMP, HLA loss does not hinder the TMMPs from killing cancer cells.
Carcinomas that can treated by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.
Sarcomas that can be treated by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.
Other solid tumors that can be treated by a method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Leukemias that can be amenable to therapy by a method disclosed herein include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukemias (neoplastic transformation of a multipotential hematopoietic stem cell or a hematopoietic cell of restricted lineage potential; c) chronic lymphocytic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemias (characterized by accumulation of lymphoblasts). Lymphomas that can be treated using a subject method include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin's lymphoma, and the like.
Other cancers that can be treated according to the methods disclosed herein include atypical meningioma, islet cell carcinoma, medullary carcinoma of the thyroid, mesenchymoma, hepatocellular carcinoma, hepatoblastoma, clear cell carcinoma of the kidney, and neurofibroma mediastinum.
Where a TMMP comprises: i) a peptide epitope that when in an MHC/peptide complex of a TMMP presents a viral epitope; and ii) a TTP that targets a cancer-associated antigen, the TMMP can be administered to an individual in need thereof to treat a cancer in the individual, where: i) the TMMP activates a T-cell that is specific for the viral epitope (e.g., a SARS-CoV-2 epitope); and ii) the cancer expresses the cancer epitope bound by the TTP. The present disclosure provides a method of treating cancer in an individual, the method comprising administering to the individual an effective amount of a TMMP, where the TMMP comprises: i) a peptide epitope that when in an MHC/peptide complex of a TMMP presents a viral epitope (e.g., a SARS-CoV-2 epitope); ii) a TTP that targets a cancer-associated antigen; and iii) a stimulatory immunomodulatory polypeptide (e.g., an IL-2 polypeptide; a 4-1BBL polypeptide; etc.).
In some cases, an “effective amount” of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual. For example, in some cases, an “effective amount” of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual by 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%, or at least 95%, compared to the number of cancer cells in the individual before administration of the TMMP, or in the absence of administration with the TMMP. In some cases, an “effective amount” of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual to undetectable levels.
In some cases, an “effective amount” of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the tumor mass/tumor volume in the individual. In some cases, an “effective amount” of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof, increases survival time of the individual. For example, in some cases, an “effective amount” of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof, increases survival time of the individual by at least 1 month, at least 2 months, at least 3 months, from 3 months to 6 months, from 6 months to 1 year, from 1 year to 2 years, from 2 years to 5 years, from 5 years to 10 years, or more than 10 years, compared to the expected survival time of the individual in the absence of administration with the TMMP.
In some cases, an “effective amount” of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof, either as a monotherapy or as part of a combination therapy, reduces the overall tumor burden in the individual, i.e., the amount of cancer in the body, or alternatively, causes the total tumor burden in the patient to remain relatively stable for a sufficient period of time for the patient to have a confirmed “stable disease” as determined by standard RECIST criteria. See, e.g., Aykan and Ozatli (2020) World J. Clin. Oncol. 11:53.
In some cases, an effective amount of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof, either as a monotherapy or as part of a combination therapy, causes the tumor size to be reduced by a sufficient amount, and for a sufficient period of time, for the patient to have a confirmed “partial response” as determined by standard RECIST criteria.
In some cases, an effective amount of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof (e.g., an individual having a tumor), either as a monotherapy or as part of a combination therapy, causes the tumor size to be reduced by a sufficient amount, and for a sufficient period of time, for the patient to have a confirmed “complete response” as determined by standard RECIST criteria.
As noted above, in some cases, in carrying out a subject treatment method, a TMMP is administered to an individual in need thereof, as the TMMP per se. In other instances, in carrying out a subject treatment method, one or more nucleic acids comprising nucleotide sequences encoding a TMMP is/are administering to an individual in need thereof. Thus, in other instances, one or more nucleic acids, e.g., one or more recombinant expression vectors, is/are administered to an individual in need thereof.
In some cases, a SARS-CoV-2 vaccine (e.g., the Moderna, Pfizer or J&J vaccine) is administered to the patient several days (e.g., 3-14 days or 7-10 days) prior to administering the TMMP in order to increase the percentage of CD8+ T cells in the patient that recognize the SARS CoV-2 peptide in the TMMP. Alternatively, prior to administering the TMMP, one or more doses of a TMMP that does not comprise a TTP can be administered to the patient, in order to prime and activate and/or induce proliferation of (and thereby increase the number of) CD8+ T cells that recognize the desired SARS CoV-2 peptide in the TMMP comprising the TTP. Alternatively (or additionally), prior to administering the TMMP, one or more doses of a T cell modulatory polypeptide can be administered to the patient, in order to prime and activate and/or induce proliferation of (and thereby increase the number of) CD8+ T cells that recognize the desired SARS CoV-2 peptide in the TMMP comprising the TTP. See, e.g., the T cell modulatory polypeptides described in WO2021/195108, published Sep. 30, 2021 (Cue Biopharma, Inc.), WO 2021/195411, published Sep. 30, 2021 (Cue Biopharma, Inc.), and WO 2022/015880, published Jan. 20, 2022 (Cue Biopharma, Inc.).
A suitable dosage can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular polypeptide or nucleic acid to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently. A TMMP of the present disclosure may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose, or higher, e.g. from 0.1 mg/kg body weight to 10 mg/kg body weight, e.g. from 1 mg/kg body weight to 5 mg/kg or from 5 mg/kg body weight to 10 mg/kg body weight; from 10-15 mg/kg, or higher, however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it can also be in the range of 1 μg to 10 mg per kilogram of body weight per minute. Generally speaking, a TMMP of the present disclosure can be administered in an amount of from about 1 mg/kg body weight to 20 mg/kg body weight, e.g., from about 1 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, or from about 15 mg/kg body weight to about 20 mg/kg body weight. Typical ranges may be from 1 mg/kg body weight to 5 mg/kg body weight or from 5 mg/kg body weight to about 10 mg/kg body weight, e.g., 1, 2, 4, 5, 6, 7 or 8 mg/kg body weight.
Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein a TMMP is administered in maintenance doses in the above ranges.
Those of skill will readily appreciate that dose levels can vary as a function of the specific TMMP, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
The frequency of administration of a TMMP can vary depending on any of a variety of factors, but generally speaking will be administered once a week, once every two weeks, once every three weeks, once every four weeks, once per month, or less frequently than once per month, e.g., once every five weeks, once every six weeks, once every two months, once every three months, etc., but also can be administered more frequently than once per week, e.g., twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), or daily (qd). In some cases, the TMMP is administered once every three weeks. Administration generally should be stopped upon disease progression or unacceptable toxicity.
The duration of administration of a TMMP can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, a TMMP can be administered over a period of time ranging from one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more. Typically, the TMMP will continue to be dosed for at least as long as the patient continues to receive a clinically determined benefit, which likely will be from at least many months to multiple years.
In some cases, a method of the present disclosure for treating cancer in an individual comprises: a) administering a TMMP; and b) administering at least one additional therapeutic agent or therapeutic treatment. Suitable additional therapeutic agents include, but are not limited to, a small molecule cancer chemotherapeutic agent, and an immune checkpoint inhibitor. Suitable additional therapeutic treatments include, e.g., radiation, surgery (e.g., surgical resection of a tumor), and the like.
A treatment method of the present disclosure can comprise co-administration of a TMMP and at least one additional therapeutic agent. By “co-administration” is meant that both a TMMP and at least one additional therapeutic agent are administered to an individual, although not necessarily at the same time, in order to achieve a therapeutic effect that is the result of having administered both the TMMP and the at least one additional therapeutic agent. The administration of the TMMP and the at least one additional therapeutic agent can be substantially simultaneous, e.g., the TMMP can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of administration of the at least one additional therapeutic agent. In some cases, a TMMP of the present disclosure is administered to an individual who is undergoing treatment with, or who has undergone treatment with, the at least one additional therapeutic agent. The administration of the TMMP can occur at different times and/or at different frequencies.
As an example, a treatment method of the present disclosure can comprise co-administration of a TMMP and an immune checkpoint inhibitor such as an antibody specific for an immune checkpoint. By “co-administration” is meant that both a TMMP and an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) are administered to an individual, although not necessarily at the same time, in order to achieve a therapeutic effect that is the result of having administered both the TMMP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). The administration of the TMMP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) can be substantially simultaneous, e.g., the TMMP can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, within about 24 hours, within 1 week, 3 weeks 3 weeks, four weeks or a month following administration of the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). The TMMP In some cases, a TMMP of the present disclosure is administered to an individual who is undergoing treatment with, or who has undergone treatment with, an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). The administration of the TMMP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) can occur at different times and/or at different frequencies. Where there is an established dosing interval for the checkpoint inhibitor, depending on the interval, it may be possible to administer the TMMP on the same day as the checkpoint inhibitor. For example, in some cases, where the dosing schedule for pembrolizumab is once every three weeks, the pharmaceutical composition comprising the TMMP may be administered on the same day. Patients who receive such combination treatment can have any CPS score (e.g., from 0 to 20, up to 30, up to 50, or greater than 50), including patients who have a CPS score of 20 or less (e.g., 15 or below or 10 or below), which may be generally indicative of a patient who is unlikely to benefit from receiving an anti-PD1 checkpoint inhibitor alone. In some cases, the combination of the TMMP is given to patients whose CPS score is 20 or less.
Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122 and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT and VISTA.
In some cases, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint polypeptide. In some cases, the anti-immune checkpoint antibody is a monoclonal antibody. In some cases, the anti-immune checkpoint antibody is humanized, or de-immunized such that the antibody does not substantially elicit an immune response in a human. In some cases, the anti-immune checkpoint antibody is a humanized monoclonal antibody. In some cases, the anti-immune checkpoint antibody is a de-immunized monoclonal antibody. In some cases, the anti-immune checkpoint antibody is a fully human monoclonal antibody. In some cases, the anti-immune checkpoint antibody inhibits binding of the immune checkpoint polypeptide to a ligand for the immune checkpoint polypeptide. In some cases, the anti-immune checkpoint antibody inhibits binding of the immune checkpoint polypeptide to a receptor for the immune checkpoint polypeptide.
Suitable anti-immune checkpoint antibodies include, but are not limited to, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline/Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex, Inc./Bristol Myer Squibb), MEDI-4736 (Medimmune/AstraZeneca), arelumab (Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep S.A.), MGA271 (Macrogenics), BMS-986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb), PF-05082566 (Pfizer), IPH2101 (Innate Pharma/Bristol-Myers Squibb), MEDI-6469 (Medlmmune/AZ), CP-870,893 (Genentech), Mogamulizumab (Kyowa Hakko Kirin), Varlilumab (CelIDex Therapeutics), Avelumab (EMD Serono), Galiximab (Biogen Idec), AMP-514 (Amplimmune/AZ), AUNP 12 (Aurigene and Pierre Fabre), Indoximod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte); KN035; and combinations thereof. For example, in some cases, the immune checkpoint inhibitor is an anti-PD-1 antibody. Suitable anti-PD-1 antibodies include, e.g., nivolumab, pembrolizumab (also known as MK-3475), cemiplimab (Libtayo®), pidilizumab, SHR-1210, PDR001, and AMP-224. In some cases, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab or PDR001. In some cases, the anti-PD-1 monoclonal antibody is cemiplimab. Suitable anti-PD1 antibodies are described in U.S. Patent Publication No. 2017/0044259. For pidilizumab, see, e.g., Rosenblatt et al. (2011) J. Immunother. 34:409-18. In some cases, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some cases, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. For tremelimumab, see, e.g., Ribas et al. (2013) J. Clin. Oncol. 31:616-22. In some cases, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some cases, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab, see, e.g., WO 2011/066389. For atezolizumab, see, e.g., U.S. Pat. No. 8,217,149. In some cases, the immune checkpoint inhibitor is an anti-TIGIT antibody that binds to T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT). In some cases, the anti-TIGIT antibody is BMS-986207 (Bristol-Myers Squibb). In some cases, the anti-TIGIT antibody is tiragolumab. In some cases, the anti-TIGIT antibody is EOS88448 (EOS-448). See, e.g., U.S. Pat. Nos. 11,008,390 and 10,189,902; U.S. Patent Publication No. 2017/0088613; and WO 2019/137541.
Among such checkpoint inhibitors, antibodies to PD-1, PD-L1 and CTLA-4 are the most common, with at least nivolumab, tremelimumab, pembrolizumab, ipilimumab, cemiplimab, atezolizumab, avelumab, tisleizumab and durvalumab having been approved by the FDA and/or regulatory agencies outside of the U.S. A TMMP of this disclosure also may be co-administered with combinations of checkpoint inhibitors, e.g., a combination of (i) an antibody to PD-1 or PD-L1, and (ii) an antibody to CTLA-4.
Subjects suitable for treatment with a method include individuals who have cancer, including individuals who have been diagnosed as having cancer, individuals who have been treated for cancer but who failed to respond to the treatment, and individuals who have been treated for cancer and who initially responded but subsequently became refractory to the treatment and/or whose disease progressed while on the prior treatment. Such subjects can include individuals whose cancers exhibit HLA loss. Such subjects can include individuals whose CPS score is 20 or less.
In some cases, the subject is an individual who is undergoing treatment with an immune checkpoint inhibitor. In some cases, the subject is an individual who has undergone treatment with an immune checkpoint inhibitor, but whose disease has progressed despite having received such treatment. In some cases, the subject is an individual who is undergoing treatment with, or who has undergone treatment with, a cancer chemotherapeutic agent. In some cases, the subject is an individual who is preparing to undergo treatment with, is undergoing treatment with, or who has undergone treatment with, an immune checkpoint inhibitor. In some cases, the subject is an individual who is preparing to undergo treatment with, is undergoing treatment with, or who has undergone treatment with, a cancer chemotherapeutic agent, radiation treatment, surgery, and/or treatment with another therapeutic agent. In some cases, a pharmaceutical composition comprising the TMMP is administered in the adjuvant or neoadjuvant setting.
Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:
Aspect 1. A T-cell modulatory multimeric polypeptide comprising: at least one heterodimer comprising: a) a first polypeptide comprising: i) a SARS-CoV-2 peptide, wherein the SARS-CoV-2 peptide has a length of from about 4 amino acids to about 25 amino acids; and ii) first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2 microglobulin (β2M) polypeptide; b) a second polypeptide comprising a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide; c) at least one immunomodulatory polypeptide, wherein the first and/or the second polypeptide comprises the at least one immunomodulatory polypeptide; d) an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, wherein the first or the second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and e) a tumor-targeting polypeptide (TTP), wherein the first and/or the second polypeptide comprises the TTP, optionally wherein one or more of the individual components of the first and/or second polypeptide in each of the above TMMP combinations are linked together by one or more linkers.
Aspect 2. A TMMP of aspect 1, wherein
Aspect 3. A TMMP of aspect 1, wherein
Aspect 4. A TMMP of any one of aspects 1-3, wherein
Aspect 5. A TMMP of any one of aspects 1-4, 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.
Aspect 6. A TMMP of aspect 5, wherein at least one of the one or more immunomodulatory polypeptides is a variant IL-2 polypeptide comprising: a) H16A and F42A substitutions; or b) H16T and F42A substitutions.
Aspect 7. A TMMP of any one of aspects 1-6, wherein the second polypeptide comprises an Ig Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide.
Aspect 8. A TMMP of aspect 7, wherein the IgG1 Fc comprises one or more amino acid substitutions that reduce or substantially eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
Aspect 9. A TMMP of aspect 8, wherein IgG1 Fc polypeptide comprises L234A and L235A substitutions.
Aspect 10. A TMMP of aspect 9, wherein at least one of the one or more linkers comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10.
Aspect 11. A TMMP of any one of aspects 1-10, wherein the TMMP comprises at least two immunomodulatory polypeptides, and wherein at least two of the immunomodulatory polypeptides are the same.
Aspect 12. A TMMP of aspect 11, wherein the two or more immunomodulatory polypeptides are in tandem.
Aspect 13. A TMMP of aspect 12, wherein the two or more immunomodulatory polypeptides are separated by a linker.
Aspect 14. A TMMP of any one of aspects 1-13, wherein the TTP is a single-chain Fv (scFv) antibody or a nanobody.
Aspect 15. A TMMP of any one of aspects 1-14, wherein the TTP binds an antigen selected from mesothelin, TROP-2, Her2, CD19, WT1, MUC1, BCMA, PSMA, B7-H3, CEACAM, CD20, CD22, CD30, CD38, CD138, and a claudin polypeptide.
Aspect 16. A TMMP of any one of aspects 1-15, wherein the TTP binds a CD19 antigen.
Aspect 17. A TMMP of any one of aspects 1-16, wherein the SARS-CoV-2 peptide has a length of from about 8 amino acids to about 12 amino acids.
Aspect 18. A TMMP of any one of aspects 1-17, wherein the SARS-CoV-2 peptide comprises from 4 to 25 contiguous amino acids of a SARS-CoV-2 surface glycoprotein.
Aspect 19. A TMMP of aspect 18, wherein the SARS-CoV-2 peptide is a surface glycoprotein peptide depicted in
Aspect 20. A TMMP any one of aspects 1-19, wherein the SARS-CoV-2 peptide has the sequence YLQPRTFLL (SEQ ID NO:218) and has a length of 9 amino acids.
Aspect 21. A TMMP of any one of aspects 1-19, wherein the first polypeptide and the second polypeptide are covalently linked to one another via one or more disulfide bonds.
Aspect 22. A TMMP of aspect 21, wherein the β2M polypeptide and the MHC heavy chain polypeptide are joined by a disulfide bond that joins a Cys residue in the β2M polypeptide and a Cys residue in the MHC heavy chain polypeptide.
Aspect 23. A TMMP of aspect 22, wherein a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide.
Aspect 24. A TMMP of aspect 21, wherein the first polypeptide chain comprises a linker between the peptide epitope and the β2M polypeptide, and wherein the disulfide bond links a Cys present in the linker with a Cys at position 84 of the MHC heavy chain polypeptide.
Aspect 25. A TMMP of any one of aspects 1-24, wherein the first and the second polypeptides are covalently linked to one another via at least 2 disulfide bonds.
Aspect 26. A TMMP of aspect 25, wherein:
Aspect 27. A TMMP of aspect 26, wherein the linker comprises the amino acid sequence GCGGS (SEQ ID NO:977).
Aspect 28. A TMMP of aspect 27, wherein the linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:978), where n is an integer from 1 to 10.
Aspect 29. A TMMP of any one of aspects 1-28, wherein the MHC class I heavy chain is an HLA-E allele heavy chain polypeptide.
Aspect 30. A TMMP of any one of aspects 1-29, wherein the second polypeptide comprises a linker between the TTP and the MHC class I heavy chain, wherein the linker has a length of less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, less than about 10 amino acids, less than 8 amino acids, or less than 6 amino acids.
Aspect 31. A TMMP of aspect 30, wherein the linker has the amino acid sequence GGGGS (SEQ ID NO:873).
Aspect 32. A homodimer comprising two heterodimers according to any one of aspects 1-31, wherein the two heterodimers are covalently linked to one another by one or more disulfide bonds between Ig Fc polypeptides present in the first and second heterodimers.
Aspect 33. A pharmaceutical composition comprising a homodimer of aspect 32.
Aspect 34. A nucleic acid comprising a nucleotide sequence encoding a first and/or second polypeptide according to any one of aspects 1-31.
Aspect 35. An expression vector comprising the nucleic acid of aspect 34.
Aspect 36. A method of selectively modulating the activity of T cell specific for an epitope, the method comprising contacting the T cell with a T-cell modulatory multimeric polypeptide according to any one of aspects 1-31 or a homodimer according to aspect 32, wherein said contacting selectively modulates the activity of the epitope-specific T cell.
Aspect 37. 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 aspects 1-31 or a pharmaceutical composition according to aspect 33.
Aspect 38. The method of aspect 37, wherein the cancer is a carcinoma, sarcoma, melanoma, leukemia, or lymphoma.
Aspect 39. The method of aspect 37 or 38, wherein said administering is intramuscular, intravenous, peritumoral, or intratumoral.
Aspect 40. The method of any one of aspects 37-39, wherein the method comprises administering a SARS-CoV-2 vaccine to the patient prior to administering to the patient an effective amount of a pharmaceutical composition comprising T-cell modulatory multimeric polypeptide.
Aspect 41. The method of any one of aspects 37-39, comprising co-administering an immune checkpoint inhibitor to the patient, optionally wherein the immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, or CTLA4.
Aspect 42. A protein comprising: at least one heterodimer comprising: a) a first polypeptide comprising: i) a SARS-CoV-2 peptide, wherein the SARS-CoV-2 peptide has a length of from about 4 amino acids to about 25 amino acids; and ii) first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2 microglobulin (β2M) polypeptide; b) a second polypeptide comprising a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide; c) an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, wherein the first or the second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and e) a tumor-targeting polypeptide (TTP), wherein the first and/or the second polypeptide comprises the TTP, optionally wherein one or more of the individual components of the first and/or second polypeptide in each of the above combinations are linked together by one or more linkers.
Aspect 43. A homodimer comprising two heterodimers according to aspect 42, wherein the two heterodimers are covalently linked to one another by one or more disulfide bonds between Ig Fc polypeptides present in the first and second heterodimers.
Aspect 44. A T-cell modulatory multimeric polypeptide (TMMP) comprising:
Aspect 45. A TMMP of aspect 44, wherein
Aspect 46. A TMMP of aspect 44 or aspect 45, wherein
Aspect 47. A TMMP of any one of aspects 44-46, wherein the second polypeptide comprises an Ig Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide.
Aspect 48. A TMMP of aspect 47, wherein the IgG1 Fc comprises one or more amino acid substitutions that reduce or substantially eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
Aspect 49. A TMMP of aspect 48, wherein IgG1 Fc polypeptide comprises L234A and L235A substitutions, wherein L234 and L235 correspond to amino acids 14 and 15 of the amino acid sequence depicted in
Aspect 50. A TMMP of aspect 46, wherein at least one of the one or more linkers comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10.
Aspect 51. A TMMP of any one of aspects 44-50, wherein the TTP is a single-chain Fv (scFv) antibody or a nanobody.
Aspect 52. A TMMP of any one of aspects 44-51, wherein the TTP binds an antigen selected from mesothelin, TROP-2, Her2, CD19, WT1, MUC1, BCMA, PSMA, B7-H3, CEACAM, CD20, CD22, CD30, CD38, CD138, and a claudin polypeptide.
Aspect 53. A TMMP of any one of aspects 44-52, wherein the TTP binds a CD19 antigen.
Aspect 54. A TMMP of any one of aspects 44-53, wherein the peptide epitope has a length of from 8 amino acids to 14 amino acids, optionally from 9 amino acids to 12 amino acids.
Aspect 55. A TMMP of any one of aspects 44-54, wherein the peptide epitope comprises from 4 to 25 contiguous amino acids of a SARS-CoV-2 surface glycoprotein.
Aspect 56. A TMMP of aspect 55, wherein the SARS-CoV-2 peptide is a surface glycoprotein peptide depicted in
Aspect 57. A TMMP any one of aspects 44-54, wherein the peptide epitope is a SARS-CoV-2 peptide epitope, and wherein the SARS-CoV-2 peptide has an amino acid sequence selected from YLQPRTFLL (SEQ ID NO:218), YLQPRTLFL (SEQ ID NO:887), YLQPRTLIL (SEQ ID NO:1201), and YLQPRTLVL (SEQ ID NO:1202), wherein the SARS-CoV-2 peptide has a length of 9 amino acids.
Aspect 58. A TMMP of any one of aspects 44-54, wherein the peptide epitope is a SARS-CoV-2 peptide epitope, and wherein the SARS-CoV-2 polypeptide is a non-structural polypeptide, optionally wherein the non-structural polypeptide is NSP13.
Aspect 59. A TMMP of aspect 58, wherein the peptide epitope is a SARS-CoV-2 peptide epitope, and wherein the SARS-CoV-2 peptide has the amino acid sequence VMPLSAPTL (SEQ ID NO:914) and has a length of 9 amino acids.
Aspect 60. A TMMP of any one of aspects 44-54, wherein the peptide epitope is an epitope of a viral antigen other than a SARS-CoV-2 epitope, optionally where in the peptide epitope is a viral epitope of a virus selected from the group of Adenoviruses, Adeno-associated virus, Alphaviruses (Togaviruses), Eastern equine encephalitis virus, Eastern equine encephalomyelitis virus, Venezuelan equine encephalomyelitis vaccine strain TC-83, Western equine encephalomyelitis virus, Arenaviruses, Lymphocytic choriomeningitis virus (non-neurotropic strains), Tacaribe virus complex, Bunyaviruses, Bunyamwera virus, Rift Valley fever virus vaccine strain MP-12, Chikungunya virus, Calciviruses, Coronaviruses, Cowpox virus, Flaviviruses (Togaviruses)—Group B Arboviruses, Dengue virus serotypes 1, 2, 3, and 4, Yellow fever virus vaccine strain 17D, Hepatitis A, B, C, D, and E viruses, the Cytomegalovirus, Epstein Barr virus, Eastern Equine encephalitis virus, Herpes simplex types 1 and 2, Herpes zoster, Human herpesvirus types 6 and 7, hepatitis C virus (HVC), hepatitis B virus (HBV), Influenza viruses types A, B, and C, Papovaviruses, Newcastle disease virus, Measles virus, Mumps virus, Parainfluenza viruses types 1, 2, 3, and 4, polyomaviruses (JC virus, BK virus), Respiratory syncytial virus, Human parvovirus (B 19), Coxsackie viruses types A and B, Echoviruses, Polioviruses, Rhinoviruses, Alastrim (Variola minor virus), Smallpox (Variola major virus), Whitepox Reoviruses, Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus), Rabies virus, Vesicular stomatitis virus, Rubivirus (rubella), Semliki Forest virus, St. Louis encephalitis virus, Venezuelan equine encephalitis virus, Venezuelan equine encephalomyelitis virus, Arenaviruses (a.k.a. South American Hemorrhagic Fever virus), Flexal, Lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantaviruses including Hantaan virus, Rift Valley fever virus, Japanese encephalitis virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus (HIV) types 1 and 2, Human T cell lymphotropic virus (HTLV) types 1 and 2, Simian immunodeficiency virus (SIV), Vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia, Crimean-Congo hemorrhagic fever virus, Ebola viruses, Marburg virus, Tick-borne encephalitis virus complex (flavi) including Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian Spring Summer encephalitis viruses, Herpesvirus simiae (Herpes B or Monkey B virus), Cercopithecine herpesvirus 1 (Herpes B virus), Equine morbillivirus (Hendra and Hendra-like viruses), Nipah virus, Variola major virus (Smallpox virus), Variola minor virus (Alastrim), African swine fever virus, African horse sickness virus, Akabane virus, Avian influenza virus (highly pathogenic), Blue tongue virus, Camel pox virus, Classical swine fever virus, Cowdria ruminantium (heartwater), Foot and mouth disease virus, Goat pox virus, Japanese encephalitis virus, Lumpy skin disease virus, Malignant catarrhal fever virus, Menangle virus, Newcastle disease virus (VVND), Vesicular stomatitis virus (exotic), and Zika virus.
Aspect 61. A TMMP of aspect 60, where the viral antigen is a cytomegalovirus (CMV) polypeptide.
Aspect 62. A T-cell modulatory multimeric polypeptide of aspect 61, wherein the CMV polypeptide is a CMV pp65 polypeptide.
Aspect 63. A T-cell modulatory multimeric polypeptide of aspect 62, wherein the peptide has the amino acid sequence NLVPMVATV (SEQ ID NO:913) and has a length of 9 amino acids.
Aspect 64. A TMMP of any one of aspects 54-63, wherein the first polypeptide and the second polypeptide are covalently linked to one another via one or more disulfide bonds.
Aspect 65. A TMMP of aspect 64, wherein the β2M polypeptide and the MHC heavy chain polypeptide are joined by a disulfide bond that joins a Cys residue in the β2M polypeptide and a Cys residue in the MHC heavy chain polypeptide.
Aspect 66. A TMMP of aspect 65, wherein a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide.
Aspect 67. A TMMP of aspect 64, wherein the first polypeptide chain comprises a linker between the peptide epitope and the β2M polypeptide, and wherein the disulfide bond links a Cys present in the linker with a Cys at position 84 of the MHC heavy chain polypeptide.
Aspect 68. A TMMP of any one of aspects 44-67, wherein the first and the second polypeptides are covalently linked to one another via at least 2 disulfide bonds.
Aspect 69. A TMMP of aspect 68, wherein:
Aspect 70. A TMMP of aspect 69, wherein the linker comprises the amino acid sequence GCGGS (SEQ ID NO:977).
Aspect 71. A TMMP of aspect 70, wherein the linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:978), where n is an integer from 1 to 10.
Aspect 72. A TMMP of any one of aspects 44-71, wherein the MHC class I heavy chain is an HLA-E allele heavy chain polypeptide.
Aspect 73. A TMMP of aspect 57, wherein:
Aspect 74. A TMMP of aspect 73, wherein:
Aspect 75. A TMMP of aspect 74, wherein:
Aspect 76. A homodimer comprising two heterodimers according to any one of aspects 44-75, wherein the two heterodimers are covalently linked to one another by one or more disulfide bonds between Ig Fc polypeptides present in the first and second heterodimers.
Aspect 77. A pharmaceutical composition comprising a heterodimer of any one of aspects 44-75 or a homodimer of aspect 76.
Aspect 78. A nucleic acid comprising a nucleotide sequence encoding a first and/or second polypeptide according to any one of aspects 44-76.
Aspect 79. An expression vector comprising the nucleic acid of aspect 78.
Aspect 80. A method of selectively modulating the activity of T cell specific for an epitope, the method comprising contacting the T cell with a T-cell modulatory multimeric polypeptide according to any one of aspects 44-75 or a homodimer according to aspect 74, wherein said contacting selectively modulates the activity of the epitope-specific T cell.
Aspect 81. 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 aspects 44-75 or a homodimer according to aspect 76.
Aspect 82. The method of aspect 81, wherein the cancer is a carcinoma, sarcoma, melanoma, leukemia, or lymphoma.
Aspect 83. The method of aspect 81 or 82, wherein said administering is intramuscular, intravenous, peritumoral, or intratumoral.
Aspect 84. The method of any one of aspects 81-83, wherein the method comprises administering a SARS-CoV-2 vaccine to the patient prior to administering to the patient an effective amount of a pharmaceutical composition comprising T-cell modulatory multimeric polypeptide.
Aspect 85. The method of any one of aspects 81-84, comprising co-administering an immune checkpoint inhibitor to the patient, optionally wherein the immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, or CTLA4.
Aspect 86. The method of aspect 85, wherein the checkpoint inhibitor is an antibody specific for PD-1, and the patient has a CPS score of 20 or less.
Aspect 87. The method of aspect 81, wherein cancer is a hematological cancer.
Aspect 88. The method of any one of aspects 81-88, wherein the cancer exhibits HLA loss.
Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:
Aspect 1. A T-cell modulatory multimeric polypeptide (TMMP) comprising:
Aspect 2. A TMMP of aspect 1, wherein
Aspect 3. A TMMP of aspect 1, wherein
Aspect 4. A TMMP of any one of aspects 1-3, wherein
Aspect 5. A TMMP of any one of aspects 1-4, 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.
Aspect 6. A TMMP of aspect 5, wherein at least one of the one or more immunomodulatory polypeptides is a variant IL-2 polypeptide, wherein the variant IL-2 polypeptide binds to IL-2Rα and IL-2RP, and wherein the IL-2 variant exhibits decreased binding to IL-2Rα and decreased binding to IL-2RP, optionally wherein the variant IL-2 comprises: a) H16A and F42A substitutions; or b) H16T and F42A substitutions, and optionally wherein the one or more immunomodulatory polypeptides is a single copy of a variant IL-2 polypeptide.
Aspect 7. A TMMP of any one of aspects 1-6, wherein the second polypeptide comprises an Ig Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide.
Aspect 8. A TMMP of aspect 7, wherein the IgG1 Fc comprises one or more amino acid substitutions that reduce or substantially eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
Aspect 9. A TMMP of aspect 8, wherein IgG1 Fc polypeptide comprises L234A and L235A substitutions, wherein L234 and L235 correspond to amino acids 14 and 15 of the amino acid sequence depicted in
Aspect 10. A TMMP of aspect 9, wherein at least one of the one or more linkers comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10.
Aspect 11. A TMMP of any one of aspects 1-10, wherein the TMMP comprises at least two immunomodulatory polypeptides, and wherein at least two of the immunomodulatory polypeptides are the same.
Aspect 12. A TMMP of aspect 11, wherein the two or more immunomodulatory polypeptides are in tandem.
Aspect 13. A TMMP of aspect 12, wherein the two or more immunomodulatory polypeptides are separated by a peptide linker.
Aspect 14. A TMMP of any one of aspects 1-13, wherein the TTP is a single-chain Fv (scFv) antibody or a nanobody.
Aspect 15. A TMMP of any one of aspects 1-14, wherein the TTP binds an antigen selected from mesothelin, TROP-2, Her2, CD19, WT1, MUC1, BCMA, PSMA, B7-H3, CEACAM, CD20, CD22, CD30, CD38, CD138, and a claudin polypeptide.
Aspect 16. A TMMP of any one of aspects 1-15, wherein the TTP binds a CD19 antigen.
Aspect 17. A TMMP of any one of aspects 1-16, wherein the SARS-CoV-2 peptide or a peptide of another pathogen has a length of from 8 amino acids to 14 amino acids, optionally from 9 amino acids to 12 amino acids.
Aspect 18. A TMMP of any one of aspects 1-16, wherein the SARS-CoV-2 peptide comprises from 4 to 25 contiguous amino acids of a SARS-CoV-2 surface glycoprotein.
Aspect 19. A TMMP of aspect 18, wherein the SARS-CoV-2 peptide is a surface glycoprotein peptide depicted in
Aspect 20. A TMMP any one of aspects 1-19, wherein the SARS-CoV-2 peptide has an amino acid sequence selected from YLQPRTFLL (SEQ ID NO:218), YLQPRTLFL (SEQ ID NO:887), YLQPRTLIL (SEQ ID NO:1201), and YLQPRTLVL (SEQ ID NO:1202), wherein the SARS-CoV-2 peptide has a length of 9 amino acids.
Aspect 21. A TMMP of any one of aspects 1-19, wherein the first polypeptide and the second polypeptide are covalently linked to one another via one or more disulfide bonds.
Aspect 22. A TMMP of aspect 21, wherein the β2M polypeptide and the MHC heavy chain polypeptide are joined by a disulfide bond that joins a Cys residue in the β2M polypeptide and a Cys residue in the MHC heavy chain polypeptide.
Aspect 23. A TMMP of aspect 22, wherein a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide.
Aspect 24. A TMMP of aspect 21, wherein the first polypeptide chain comprises a peptide linker between the peptide epitope and the β2M polypeptide, and wherein the disulfide bond links a Cys present in the linker with a Cys at position 84 of the MHC heavy chain polypeptide.
Aspect 25. A TMMP of aspect 4, wherein:
Aspect 26. A TMMP of aspect 25, wherein:
Aspect 27. A TMMP of aspect 26, wherein:
Aspect 28. A TMMP of aspect 27, wherein:
Aspect 29. A TMMP of any one of aspects 1-24, wherein the first and the second polypeptides are covalently linked to one another via at least 2 disulfide bonds.
Aspect 30. A TMMP of aspect 25, wherein:
Aspect 31. A TMMP of aspect 30, wherein the linker comprises the amino acid sequence GCGGS (SEQ ID NO:977), or the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:978), where n is an integer from 1 to 10.
Aspect 32. A TMMP of any one of aspects 1-31, wherein the MHC class I heavy chain is an HLA-E allele heavy chain polypeptide.
Aspect 33. A TMMP of any one of aspects 1-32, wherein the second polypeptide comprises a linker between the TTP and the MHC class I heavy chain, wherein the peptide linker has a length of less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, less than about 10 amino acids, less than about 8 amino acids, or less than about 6 amino acids.
Aspect 34. A TMMP of any one of aspects 4-32, wherein the second polypeptide comprises a linker between the TTP and the MHC class I heavy chain, wherein the peptide linker comprises no more than about 12 amino acids, e.g., no more than 10 amino acids in length (e.g., a (GGGGS)2 (SEQ ID NO:947) linker), no more than 8 amino acids in length, no more than 7 amino acids in length, no more than 6 amino acids in length, or no more than five amino acids in length, (e.g., a GGGGS (SEQ ID NO:873) linker).
Aspect 35. A TMMP of aspect 34, wherein the peptide linker is no more than five amino acids in length and optionally has the amino acid sequence GGGGS (SEQ ID NO:873), or is no more than 10 amino acids in length and optionally has the amino acid sequence (GGGGS)2 (SEQ ID NO:947).
Aspect 36. A TMMP of any one of aspects 33-35, wherein the second polypeptide comprises a peptide linker between the Ig Fc polypeptide and one of the at least one immunomodulatory polypeptides, and wherein the peptide linker between the Ig Fc polypeptide and the immunomodulatory polypeptide is (GGGGS)3 (SEQ ID NO:875) or (AP)4 (SEQ ID NO:963).
Aspect 37. A TMMP of aspect 1, wherein:
Aspect 38. A TMMP of aspect 1, wherein:
Aspect 39. A TMMP of aspect 1, wherein:
Aspect 40. A homodimer comprising two heterodimers according to any one of aspects 1-39, wherein the two heterodimers are covalently linked to one another by one or more disulfide bonds between Ig Fc polypeptides present in the first and second heterodimers.
Aspect 41. A pharmaceutical composition comprising a heterodimer of any one of aspects 1-39 or a homodimer of aspect 40.
Aspect 42. A composition comprising:
Aspect 43. A composition comprising:
Aspect 44. A method of selectively modulating the activity of T cell specific for an epitope, the method comprising contacting the T cell with a T-cell modulatory multimeric polypeptide according to any one of aspects 1-39 or a homodimer according to aspect 40, wherein said contacting selectively modulates the activity of the epitope-specific T cell.
Aspect 45. 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 aspects 1-39 or a homodimer according to aspect 40.
Aspect 46. The method of aspect 45, wherein the cancer is a carcinoma, sarcoma, melanoma, leukemia, or lymphoma.
Aspect 47. The method of aspect 45 or 46, wherein said administering is intramuscular, intravenous, peritumoral, or intratumoral.
Aspect 48. The method of any one of aspects 45-47, wherein the method comprises administering a SARS-CoV-2 vaccine to the patient prior to administering to the patient an effective amount of a pharmaceutical composition comprising T-cell modulatory multimeric polypeptide.
Aspect 49. The method of any one of aspects 45-48, comprising co-administering an immune checkpoint inhibitor to the patient, optionally wherein the immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, or CTLA4.
Aspect 50. A T-cell modulatory multimeric polypeptide (TMMP) comprising:
Aspect 51. A TMMP of aspect 50, wherein
Aspect 52. A TMMP of aspect 50 or aspect 51, wherein
Aspect 53. A TMMP of any one of aspects 50-52, wherein the second polypeptide comprises an Ig Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide.
Aspect 54. A TMMP of aspect 53, wherein the IgG1 Fc comprises one or more amino acid substitutions that reduce or substantially eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
Aspect 55. A TMMP of aspect 54, wherein IgG1 Fc polypeptide comprises L234A and L235A substitutions, wherein L234 and L235 correspond to amino acids 14 and 15 of the amino acid sequence depicted in
Aspect 56. A TMMP of aspect 52, wherein at least one of the one or more linkers comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10.
Aspect 57. A TMMP of any one of aspects 50-56, wherein the TTP is a single-chain Fv (scFv) antibody or a nanobody.
Aspect 58. A TMMP of any one of aspects 50-57, wherein the TTP binds an antigen selected from mesothelin, TROP-2, Her2, CD19, WT1, MUC1, BCMA, PSMA, B7-H3, CEACAM, CD20, CD22, CD30, CD38, CD138, and a claudin polypeptide.
Aspect 59. A TMMP of any one of aspects 50-58, wherein the TTP binds a CD19 antigen.
Aspect 60. A TMMP of any one of aspects 50-59, wherein the peptide epitope has a length of from 8 amino acids to 14 amino acids, optionally from 9 amino acids to 12 amino acids.
Aspect 61. A TMMP of any one of aspects 50-60, wherein the peptide epitope comprises from 4 to 25 contiguous amino acids of a SARS-CoV-2 surface glycoprotein.
Aspect 62. A TMMP of aspect 61, wherein the SARS-CoV-2 peptide is a surface glycoprotein peptide depicted in
Aspect 63. A TMMP any one of aspects 50-60, wherein the peptide epitope is a SARS-CoV-2 peptide epitope, and wherein the SARS-CoV-2 peptide has an amino acid sequence selected from YLQPRTFLL (SEQ ID NO:218), YLQPRTLFL (SEQ ID NO:887), YLQPRTLIL (SEQ ID NO:1201), and YLQPRTLVL (SEQ ID NO:1202), wherein the SARS-CoV-2 peptide has a length of 9 amino acids.
Aspect 64. A TMMP of any one of aspects 50-60, wherein the peptide epitope is a SARS-CoV-2 peptide epitope, and wherein the SARS-CoV-2 polypeptide is a non-structural polypeptide, optionally wherein the non-structural polypeptide is NSP13.
Aspect 65. A TMMP of aspect 64, wherein the peptide epitope is a SARS-CoV-2 peptide epitope, and wherein the SARS-CoV-2 peptide has the amino acid sequence VMPLSAPTL (SEQ ID NO:914) and has a length of 9 amino acids.
Aspect 66. A TMMP of any one of aspects 1-60, wherein the peptide epitope is an epitope of a pathogen other than a SARS-CoV-2 epitope, optionally where in the peptide epitope is a viral epitope of a virus selected from the group of Adenoviruses, Adeno-associated virus, Alphaviruses (Togaviruses), Eastern equine encephalitis virus, Eastern equine encephalomyelitis virus, Venezuelan equine encephalomyelitis vaccine strain TC-83, Western equine encephalomyelitis virus, Arenaviruses, Lymphocytic choriomeningitis virus (non-neurotropic strains), Tacaribe virus complex, Bunyaviruses, Bunyamwera virus, Rift Valley fever virus vaccine strain MP-12, Chikungunya virus, Calciviruses, Coronaviruses, Cowpox virus, Flaviviruses (Togaviruses)—Group B Arboviruses, Dengue virus serotypes 1, 2, 3, and 4, Yellow fever virus vaccine strain 17D, Hepatitis A, B, C, D, and E viruses, the Cytomegalovirus, Epstein Barr virus, Eastern Equine encephalitis virus, Herpes simplex types 1 and 2, Herpes zoster, Human herpesvirus types 6 and 7, hepatitis C virus (HVC), hepatitis B virus (HBV), Influenza viruses types A, B, and C, Papovaviruses, Newcastle disease virus, Measles virus, Mumps virus, Parainfluenza viruses types 1, 2, 3, and 4, polyomaviruses (JC virus, BK virus), Respiratory syncytial virus, Human parvovirus (B 19), Coxsackie viruses types A and B, Echoviruses, Polioviruses, Rhinoviruses, Alastrim (Variola minor virus), Smallpox (Variola major virus), Whitepox Reoviruses, Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus), Rabies virus, Vesicular stomatitis virus, Rubivirus (rubella), Semliki Forest virus, St. Louis encephalitis virus, Venezuelan equine encephalitis virus, Venezuelan equine encephalomyelitis virus, Arenaviruses (a.k.a. South American Hemorrhagic Fever virus), Flexal, Lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantaviruses including Hantaan virus, Rift Valley fever virus, Japanese encephalitis virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus (HIV) types 1 and 2, Human T cell lymphotropic virus (HTLV) types 1 and 2, Simian immunodeficiency virus (SIV), Vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia, Crimean-Congo hemorrhagic fever virus, Ebola viruses, Marburg virus, Tick-borne encephalitis virus complex (flavi) including Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian Spring Summer encephalitis viruses, Herpesvirus simiae (Herpes B or Monkey B virus), Cercopithecine herpesvirus 1 (Herpes B virus), Equine morbillivirus (Hendra and Hendra-like viruses), Nipah virus, Variola major virus (Smallpox virus), Variola minor virus (Alastrim), African swine fever virus, African horse sickness virus, Akabane virus, Avian influenza virus (highly pathogenic), Blue tongue virus, Camel pox virus, Classical swine fever virus, Cowdria ruminantium (heartwater), Foot and mouth disease virus, Goat pox virus, Japanese encephalitis virus, Lumpy skin disease virus, Malignant catarrhal fever virus, Menangle virus, Newcastle disease virus (VVND), Vesicular stomatitis virus (exotic), and Zika virus.
Aspect 67. A TMMP of aspect 66, where the viral antigen is a cytomegalovirus (CMV) polypeptide.
Aspect 68. A T-cell modulatory multimeric polypeptide of aspect 67, wherein the CMV polypeptide is a CMV pp65 polypeptide.
Aspect 69. A T-cell modulatory multimeric polypeptide of aspect 68, wherein the peptide has the amino acid sequence NLVPMVATV (SEQ ID NO:913) and has a length of 9 amino acids.
Aspect 70. A TMMP of any one of aspects 60-69, wherein the first polypeptide and the second polypeptide are covalently linked to one another via one or more disulfide bonds.
Aspect 71. A TMMP of aspect 70, wherein the β2M polypeptide and the MHC heavy chain polypeptide are joined by a disulfide bond that joins a Cys residue in the β2M polypeptide and a Cys residue in the MHC heavy chain polypeptide.
Aspect 72. A TMMP of aspect 71, wherein a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide.
Aspect 73. A TMMP of aspect 70, wherein the first polypeptide chain comprises a linker between the peptide epitope and the β2M polypeptide, and wherein the disulfide bond links a Cys present in the linker with a Cys at position 84 of the MHC heavy chain polypeptide.
Aspect 74. A TMMP of any one of aspects 50-73, wherein the first and the second polypeptides are covalently linked to one another via at least 2 disulfide bonds.
Aspect 75. A TMMP of aspect 74, wherein:
Aspect 76. A TMMP of aspect 75, wherein the linker comprises the amino acid sequence GCGGS (SEQ ID NO:977) or amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:978), where n is an integer from 1 to 10.
Aspect 77. A TMMP of any one of aspects 50-76, wherein the MHC class I heavy chain is an HLA-E allele heavy chain polypeptide.
Aspect 78. A TMMP of any one of aspects 52-77, wherein the peptide linker is no more than five amino acids in length and optionally has the amino acid sequence GGGGS (SEQ ID NO:873), or is no more than 10 amino acids in length and optionally has the amino acid sequence (GGGGS)2 (SEQ ID NO:947).
Aspect 79. A TMMP of any one of aspects 52-78, wherein:
Aspect 80. A TMMP of aspect 79, wherein:
Aspect 81. A TMMP of aspect 80, wherein:
Aspect 82. A homodimer comprising two heterodimers according to any one of aspects 50-81, wherein the two heterodimers are covalently linked to one another by one or more disulfide bonds between Ig Fc polypeptides present in the first and second heterodimers.
Aspect 83. A pharmaceutical composition comprising a heterodimer of any one of aspects 50-81 or a homodimer of aspect 82.
Aspect 84. A composition comprising:
Aspect 85. A composition comprising:
Aspect 86. A method of selectively modulating the activity of T cell specific for an epitope, the method comprising contacting the T cell with a T-cell modulatory multimeric polypeptide according to any one of aspects 50-81 or a homodimer according to aspect 82, wherein said contacting selectively modulates the activity of the epitope-specific T cell.
Aspect 87. 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 aspects 50-81 or a homodimer according to aspect 82.
Aspect 88. The method of aspect 87, wherein the cancer is a carcinoma, sarcoma, melanoma, leukemia, or lymphoma.
Aspect 89. The method of aspect 87 or 88, wherein said administering is intramuscular, intravenous, peritumoral, or intratumoral.
Aspect 90. The method of any one of aspects 87-89, wherein the method comprises administering a SARS-CoV-2 vaccine to the patient prior to administering to the patient an effective amount of a pharmaceutical composition comprising T-cell modulatory multimeric polypeptide.
Aspect 91. The method of any one of aspects 87-90, comprising co-administering an immune checkpoint inhibitor to the patient, optionally wherein the immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, or CTLA4.
Aspect 92. The method of aspect 91, wherein the checkpoint inhibitor is an antibody specific for PD-1, and the patient has a CPS score of 20 or less.
Aspect 93. The method of aspect 87, wherein cancer is a hematological cancer.
Aspect 94. The method of any one of aspects 87-93, wherein the cancer exhibits HLA loss.
Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:
Aspect 1. A T-cell modulatory multimeric polypeptide (TMMP) comprising at least one heterodimer comprising: a) a first polypeptide comprising from N-terminus to C-terminus: i) a SARS-CoV-2 peptide, wherein the SARS-CoV-2 peptide or a peptide of another pathogen has a length of from about 4 amino acids to about 25 amino acids, ii) an optional peptide linker, and iii) first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2 microglobulin (β2M) polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) a tumor-targeting polypeptide (TTP), ii) a peptide linker, iii) a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide, iv) an optional linker, v) an immunoglobulin (Ig) Fc polypeptide, vi) an optional linker, and vii) at least one immunomodulatory polypeptide, wherein the peptide linker between the TTP and the MHC class I heavy chain polypeptide comprises no more than about 12 amino acids, e.g., no more than 10 amino acids in length (e.g., a (GGGGS)2 (SEQ ID NO:947) linker), no more than 8 amino acids in length, no more than 7 amino acids in length, no more than 6 amino acids in length, or no more than 5 amino acids in length, (e.g., a GGGGS (SEQ ID NO:873) linker).
Aspect 2. The TMMP of aspect 1, wherein the peptide linker between the TTP and the MHC class I heavy chain polypeptide is 5 amino acids in length and has the amino acid sequence GGGGS or is 10 amino acids in length and has the amino acid sequence (GGGGS)2 (SEQ ID NO:947).
Aspect 3. The TMMP of aspect 1, wherein the peptide linker between the TTP and the MHC class I heavy chain polypeptide is 5 amino acids in length and has the amino acid sequence GGGGS.
Aspect 4. The TMMP of any one of aspects 1-3, wherein the at least one immunomodulatory polypeptide is a variant IL-2 polypeptide.
Aspect 5. The TMMP of aspect 4, wherein the at least one variant IL-2 polypeptide binds to IL-2R and exhibits decreased or substantially no binding to IL-2Ra, and also exhibits decreased binding to IL-2Rβ such that the IL-2 variant polypeptide exhibits an overall reduced affinity for IL-2R, optionally wherein the at least one variant comprises substitutions at H16 and F42 selected from H16A, F42A; H16T, F42A; H16E, F42A; H16D, F42A; H16A, F42K; H16T, F42K; H16E, F42K; and H16D, F42K.
Aspect 6. The TMMP of aspect 5, wherein the at least one immunomodulatory polypeptide is a single copy of a variant IL-2 polypeptide.
Aspect 7. The TMMP of aspect 6, wherein the variant IL-2 polypeptide comprises substitutions at H16 and F42.
Aspect 8. The TMMP of aspect 7, wherein the variant IL-2 polypeptide comprises H16A and F42A substitutions.
Aspect 9. The TMMP of aspect 8, wherein the peptide linker between the TTP and the MHC class I heavy chain polypeptide has the amino acid sequence GGGGS (SEQ ID NO:873).
Aspect 10. The TMMP of aspect 9, wherein: the MHC class I heavy chain polypeptide is an HLA-A02 polypeptide and the SARS-CoV-2 peptide is YLQPRTFLL (SEQ ID NO:218); or the MHC class I heavy chain polypeptide is an HLA-E polypeptide and the SARS-CoV-2 peptide is VMPLSAPTL (SEQ ID NO:914) or YLQPRTFLL (SEQ ID NO:218).
Aspect 11. A homodimer comprising two heterodimers according to any one of aspects 1-10, wherein the two heterodimers are covalently linked to one another by one or more disulfide bonds between Ig Fc polypeptides present in the first and second heterodimers.
Aspect 12. A method of treating a patient having a cancer, the method comprising administering to the patient an effective amount of a pharmaceutical composition comprising an effective amount of a TMMP according to any one of aspects 1-10.
Aspect 13. A method of treating a patient having a cancer, the method comprising administering to the patient an effective amount of a pharmaceutical composition comprising an effective amount of a homodimer comprising two heterodimers according to any one of aspects 1-10, wherein the two heterodimers are covalently linked to one another by one or more disulfide bonds between Ig Fc polypeptides present in the first and second heterodimers.
Aspect 14. A T-cell modulatory multimeric polypeptide (TMMP) comprising at least one heterodimer comprising: a) a first polypeptide comprising: i) a SARS-CoV-2 peptide, wherein the SARS-CoV-2 peptide or a peptide of another pathogen has a length of from about 4 amino acids to about 25 amino acids, and ii) first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a f2 microglobulin (β2M) polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) a tumor-targeting polypeptide (TTP), ii) a peptide linker, iii) a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide, iv) an optional linker, and v) an immunoglobulin (Ig) Fc polypeptide, wherein the peptide linker between the TTP and the MHC class I heavy chain polypeptide comprises no more than about 12 amino acids, e.g., no more than 10 amino acids in length (e.g., a (GGGGS)2 (SEQ ID NO:947) linker), no more than 8 amino acids in length, no more than 7 amino acids in length, no more than 6 amino acids in length, or no more than five amino acids in length, (e.g., a GGGGS (SEQ ID NO:873) linker).
Aspect 15. The TMMP of aspect 14, wherein the peptide linker between the TTP and the MHC class I heavy chain polypeptide has the amino acid sequence GGGGS (SEQ ID NO:873) or (GGGGS)2 (SEQ ID NO:947).
Aspect 16. The TMMP of aspect 15, wherein the peptide linker between the TTP and the MHC class I heavy chain polypeptide has the amino acid sequence GGGGS (SEQ ID NO:873).
Aspect 17. The TMMP of any one of aspects 14-16, wherein: the MHC class I heavy chain polypeptide is an HLA-A02 polypeptide and the SARS-CoV-2 peptide is YLQPRTFLL; or the MHC class I heavy chain polypeptide is an HLA-E polypeptide and the SARS-CoV-2 peptide is VMPLSAPTL (SEQ ID NO:914) or YLQPRTFLL (SEQ ID NO:218).
Aspect 18. A homodimer comprising two heterodimers according to any one of aspects 14-17, wherein the two heterodimers are covalently linked to one another by one or more disulfide bonds between Ig Fc polypeptides present in the first and second heterodimers.
Aspect 19. A method of treating a patient having a cancer, the method comprising administering to the patient an effective amount of a pharmaceutical composition comprising an effective amount of a TMMP according to any one of aspects 14-17.
Aspect 20. A method of treating a patient having a cancer, the method comprising administering to the patient an effective amount of a pharmaceutical composition comprising an effective amount of a homodimer comprising two heterodimers according to any one of aspects 14-17, wherein the two heterodimers are covalently linked to one another by one or more disulfide bonds between Ig Fc polypeptides present in the first and second heterodimers.
Aspect 21. The method of any one of aspects 12, 13, 19 or 20, wherein the patient is further administered a checkpoint inhibitor that is an antibody specific for PD-1, and the patient has a CPS score of 20 or less.
Aspect 22. The method of any one of aspects 12, 13, 19, 20 or 21, wherein cancer is a hematological cancer.
Aspect 23. The method of any one of aspects 12, 13, 19 or 20-22, wherein the cancer exhibits HLA loss.
The following example is put forth so as to provide those of ordinary skill in the art with a more complete disclosure and is not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
Peripheral blood mononuclear cells (PBMCs), obtained from human donors (Leukopaks, designated “L” in
PBMCs from previously identified donors who were vaccinated with a SARS CoV-2 vaccine were exposed in vitro with a dose response of a protein (designated “TMP” in
PBMCs from a SARS-CoV2-responsive and cytomegalovirus (CMV)-responsive donor were incubated with the following constructs:
The PBMCs were from a donor that has at baseline has SCV2-reactive (0.05%) and CMV-reactive (0.5%) CD8 T cells in unstimulated PBMCs. The various constructs described above were added in a volume of 500 μL to PBMCs (2×106 PBMCs in 500 μL R10 medium), to a total volume of 1 mL. The PBMCs were incubated at 37° C. with the various constructs for 24 to 36 hours. After the incubation period, the PBMCs were tested by flow cytometry for: 1) activation of SARS-CoV2 (SCV2)-specific T cells present in the PBMCs, by assessing co-expression of CD127 and CD69; and 2) their ability to kill CD19-positive target B cells.
Activation of SCV2-specific T cells was tested by analyzing cell surface expression of CD137 and CD69, and by reactivity of the CD137+ and CD69+ T cells with SCV2, specifically, reactivity with the SCV2 peptide YLQPRTFLL (SEQ ID NO:218). The data are shown in
Killing of the target B cells was measured as the total number of live B cells remaining in the well after 24 hours. The data are shown in
PBMCs from a donor (“L84”) reactive to both SARS-CoV-2 and CMV were incubated with various constructs. PBMCs were cultured in a 24-well plate at 2.5×106 PBMCs/well in a total volume of 1 mL per well. Constructs were added to the wells at a final concentration of 500 nM, 50 nM, 5 nM, 0.5 nM, 0.05 nM, and 0.005 nM. The constructs were as follows:
Each of these constructs is described in Example 2; amino acid sequences of the first and second polypeptides of the constructs are provided in
The PBMCs were incubated at 37° C. with the various constructs for 24 to 36 hours. After the incubation period, the PBMCs were tested by flow cytometry for SARS-CoV-2 (SCV)-specific and CMV-specific CD8+ T cell expansion. The data are shown in
As shown in
As shown in
Constructs as described in Example 2 and Example 3 were incubated with (i) PBMCs (the same L84 PBMCs as in Examples 2 and 3) and (ii) Ramos cells (American Type Culture Collection (ATCC) No. CRL-1596), a CD19+ Burkitts lymphoma cell line. The effect of the various constructs on PBMC-mediated killing of the target Ramos cells was assessed. The results are shown in
As shown in
The effect of the linker between the scFv and the MHC class I heavy chain polypeptide, and the effect of the linker between the Ig Fc polypeptide and the MOD, on: i) the ability to induce killing of tumor cells; and ii) the ability to expand (induced proliferation of) anti-viral T cells, was assessed. Various constructs were made, which had different combinations of: i) the linker between the scFv and the MHC class I heavy chain polypeptide; and ii) the linker between the Ig Fc polypeptide and the MOD. The effect of these constructs on inducing tumor cell killing and on inducing proliferation of anti-viral T cells was determined using assays as described in Examples 2-4.
All constructs comprised: i) the SARS-CoV2 peptide YLQPRTFLL (SEQ ID NO:218); ii) an anti-CD19 scFv as the TTP, where the amino acid sequence of the anti-CD19 scFv is depicted in
Varying the Linker Between the scFv and the HLA Heavy Chain Polypeptide
The effect of the length of the linker between the scFv and the HLA heavy chain polypeptide on induction of target tumor cell killing induced by a TMMP was determined. The tumor cell target was RAMOS cells, and target tumor cell killing was assessed as described above. The linker between the scFv and the HLA heavy chain polypeptide was (GGGGS)n (SEQ ID NO:873), where n was 1, 2, 3, or 4. The constructs tested were as follows:
Construct 4518-4700. This construct has a first polypeptide designated “4700” in
Construct 4519-4700. This construct has a first polypeptide designated “4700” in
Construct 4520-4700. This construct has a first polypeptide designated “4700” in
Construct 4337-4700. This construct has a first polypeptide designated “4700” in
The features of these constructs are summarized in Table 6, below.
The data are presented in
The data show that TMMPs with shorter linkers between the scFv polypeptide and the HLA heavy chain polypeptide are more effective at inducing target tumor cell killing.
The effect of variant IL-2 valency (i.e., the number of copies of variant IL-2 polypeptide per heavy chain polypeptide) and the effect of the linker between the Ig Fc polypeptide and the variant IL-2 polypeptide, was determined. The number of variant IL-2 polypeptide was none, 1, 2, or 3. The linker between the Ig Fc polypeptide and the variant IL-2 polypeptide was either (GGGGS)3 (SEQ ID NO:875) (“(G4S)3”) or (AP)4 (SEQ ID NO:963). The constructs tested were as follows:
Construct 4656-4700. This construct is a “MOD-less” construct, and has a first polypeptide designated “4700” in
Construct 4728-4700. This construct has a first polypeptide designated “4700” in
Construct 4518-4700. This construct has a first polypeptide designated “4700” in
Construct 4729-4700. This construct has a first polypeptide designated “4700” in
Construct 4730-4700. This construct has a first polypeptide designated “4700” in
Construct 4701-4700. This construct has a first polypeptide designated “4700” in
Construct 4731-4700. This construct has a first polypeptide designated “4700” in
Construct 1715-4700. This construct has a first polypeptide designated “4700” in
Construct 1715-1717. This construct has a first polypeptide designated “1717” in
Features of these constructs are summarized in Table 7, below.
The data are presented in
For the data presented in
For the data presented in
While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope. All such modifications are intended to be within the scope of the claims appended hereto.
This application claims the benefit of U.S. Provisional Patent Application No. 63/283,028, filed Nov. 24, 2021, U.S. Provisional Patent Application No. 63/309,288, filed Feb. 11, 2022, and U.S. Provisional Patent Application No. 63/338,131, filed May 4, 2022, which applications are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63283028 | Nov 2021 | US | |
| 63309288 | Feb 2022 | US | |
| 63338131 | May 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US22/80263 | Nov 2022 | WO |
| Child | 18736261 | US |
