Patent Application
20230201335
An adaptive immune response involves the engagement of the T cell receptor (TCR), present on the surface of a T cell, with a small antigenic molecule (e.g., a 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. In addition to epitope-specific cell targeting, T cells may be targeted by immunomodulatory proteins found in, for example, APCs, that affect various functions of the target cells (e.g., activation or inhibition of various T cell functions) through of their costimulatory proteins found, for example, on the APC with counterpart costimulatory proteins (e.g., receptors) on 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, costimulatory proteins are not epitope specific, and instead are generally expressed on all T cells or on subsets of cells.
APCs generally serve to capture and break the proteins from foreign organisms, or abnormal proteins (e.g., from genetic mutation in cancer cells), into smaller fragments suitable as signals for scrutiny by the larger immune system, including T cells. In particular, APCs break down proteins into small peptide fragments, which are then paired with proteins of the major histocompatibility complex (“MHC”) and displayed on the cell surface. Cell surface display of an MHC together with a peptide fragment, also known as a T cell epitope, provides the underlying scaffold surveilled by T cells, allowing for specific recognition. The peptide fragments can be pathogen-derived (infectious agent-derived), tumor-derived, or derived from natural host proteins (self-proteins). Moreover, APCs can recognize other foreign components, such as bacterial toxins, viral proteins, viral DNA, viral RNA, etc., whose presence denotes an escalated threat level. The APCs relay this information to T cells through additional costimulatory signals in order to generate a more effective response.
T cells recognize peptide-major histocompatibility complex (“pMHC”) complexes through a specialized cell surface receptor, the T cell receptor (“TCR”). The TCR is unique to each T cell; as a consequence, each T cell is highly specific for a particular pMHC target. In order to adequately address the universe of potential threats, a very large number (10,000,000) of distinct T cells with distinct TCRs exist in the human body. Further, any given T cell, specific for a particular T cell peptide, is initially a very small fraction of the total T cell population. Although normally dormant and in limited numbers, T cells bearing specific TCRs can be readily activated and amplified by APCs to generate highly potent T cell responses that involve many millions of T cells. Such activated T cell responses are capable of attacking and clearing viral infections, bacterial infections, and other cellular threats including tumors. Conversely, the broad, non-specific activation of overly active T cell responses against self-antigens or shared antigens can give rise to T cells that inappropriately attack and destroy healthy tissues or cells.
Human MHC proteins are referred to as human leukocyte antigens (HLA) in humans. HLA proteins are divided into two major classes, Class I and Class II proteins, which are encoded by separate loci. Unless expressly stated otherwise, for the purpose of this disclosure, references to MHC or HLA Class I proteins. HLA Class I proteins each comprise a heavy chain (sometimes denoted “MHC-H”) encoded by gene loci including the classical HLA-A, HLA-B, HLA-C, and non-classical HLA-E, HLA-F and HLA-G loci. HLA Class I proteins also include a light chain protein, the β-2 microglobulin (β2M) polypeptide.
The present disclosure provides multimeric antigen-presenting polypeptide complexes (“MAPP” singular and “MAPPs” plural) that are at least heterodimeric and include at least one framework polypeptide and at least one dimerization polypeptide. Framework polypeptides comprise one or more polypeptide dimerization sequence that permits specific binding with other polypeptides (dimerization polypeptides) having a counterpart dimerization sequence thereby forming at least a heterodimer (See
The framework and dimerization peptide containing MAPPs, duplex MAPPs, and MAPPs of higher order (e.g., triplex MAPPs) described herein provide a means by which epitope-presenting peptides (“peptide epitopes” or simply “epitopes”) may be presented in the context of an MHC (e.g., HLA) to a target T cell displaying a TCR specific for the epitope, while at the same time permitting for the flexible presentation of one or more immunomodulatory polypeptides (“MODs”). The MAPPs, duplex MAPPs, and higher order MAPPs thereby permit delivery of one or more MODs in an epitope selective (e.g., dependent/specific) manner that permits (i) formation of an active immune synapse with a target T cell selective for the epitope, and (ii) modulation (e.g., control/regulation) of the target T cell's response to the epitope.
The presentation by a MAPP of a peptide epitope to a target T cell is accomplished via a moiety that comprises MHC Class I polypeptides and the peptide epitope. Such moieties may be either (i) a single polypeptide chain, or (ii) a complex comprising two or more polypeptide chains.
Where the peptide epitope, an MHC-H and β2M polypeptide sequences, and optionally one or more MODs are provided in one polypeptide, it is termed an “epitope presenting sequence” or “presenting sequence.” See, e.g.,
As an alternative to utilizing a single polypeptide to present an epitope, the MHC components (MHC-H polypeptide and β2M polypeptide sequences) and the epitope may be divided (split) into two separate polypeptide sequences, which together are denoted herein as an “epitope presenting complex” or “presenting complex.” A presenting complex is integrated into a MAPP by having a presenting complex first amino acid sequence (“presenting complex 1st sequence”) as part of a framework or dimerization polypeptide. The remaining MHC sequence(s) are part of a polypeptide termed the presenting complex second amino acid sequence (“presenting complex 2nd sequence”). The peptide epitope and any independently selected MODs that are present may be part of the polypeptide comprising either the presenting complex 1st sequence or the presenting complex 2nd sequence. The presenting complex 1st sequence and presenting complex 2nd sequence generally associate through non-covalent interactions between the MHC-H chain and the β2M polypeptide sequence, and may be stabilized by disulfide bonds between either the MHC sequences or peptide/polypeptide linkers attached to the N- or C-terminus of the MHC sequences. The presenting complex 1st sequence and presenting complex 2nd sequence may also associate through dimerization or interspecific dimerization sequences if present in those polypeptides.
Although an individual MAPP may not comprise a presenting sequence or presenting complex, for the purpose of this disclosure the MAPPs are, unless stated otherwise, understood to comprise at least one presenting sequence or presenting complex.
MAPPs that comprise a presenting sequence typically contain one or two presenting sequences. Duplex MAPPS thus typically comprise two, three or four presenting sequences, but also may comprise one presenting sequence (e.g., if one of the MAPPS does not comprise a presenting sequence). MAPPs and duplex MAPPs may comprise more presenting sequences depending on, for example, the number of dimerization sequences in the framework polypeptide. The presenting sequences may be integrated into a MAPP as part of a framework polypeptide, a dimerization polypeptide, or both. Compare, for example,
Likewise, MAPPs with presenting complexes typically contain one or two presenting complexes, and accordingly, duplex MAPPs with presenting complexes typically comprise two, three or four presenting complexes, but also may comprise one presenting complex (e.g., if one of the MAPPs does not comprise a presenting complex). As discussed above, MAPPs and duplex MAPPs may comprise more presenting complexes depending on, for example, the number of dimerization sequences in the framework polypeptide.
MAPPs and accordingly their higher order complexes (duplexes, triplexes etc.) comprising MHC Class I polypeptide sequences and a peptide epitope for presentation to a TCR, may present peptides to T cells (e.g., CD8+ T cells) that have a TCR specific for the epitope. Once engaged with the TCR of a T cell, the effect of a MAPP on the T cell depends on which MODs, if any, are present as part of the MAPP.
MOD-containing MAPPs can function as a means of selectively delivering the MODs to T cells specific for the MAPP-associated epitope, thereby resulting in MOD-driven responses to those MAPPs (e.g., proliferation, reduction in number and/or suppression of T cells specific for the MAPP-associated epitope). Depending on the chosen MOD, the incorporation of one or more MODs with increased affinity for their cognate receptor on T cells (“co-MOD”) may reduce the specificity of MAPPs and duplex MAPPs for epitope specific T cells where MOD—co-MOD binding interactions significantly compete with MHC/epitope binding to target cell TCR. Conversely, and again depending on the chosen MOD, the inclusion of MODs with reduced affinity for their co-MOD(s), and the affinity of the epitope for a TCR, may provide for enhanced selectivity of MAPPs and duplex MAPPs, while retaining the desired activity of the MODs. Where a MOD already possesses a relatively low affinity for its cognate receptor, mutations that reduce the affinity may be unnecessary and/or undesirable.
The ability of MAPPs (e.g., duplex MAPPs) to modulate T cells provides methods of modulating T cell activity in vitro and in vivo, and accordingly the use of MAPPs as therapeutics useful in methods of treating a variety of diseases and conditions including cancers, autoimmune diseases, and infectious diseases.
The present disclosure provides nucleic acids comprising nucleotide sequences and vectors encoding individual MAPP polypeptides and MAPPs (e.g., all polypeptides of a MAPP), as well as cells genetically modified with the nucleic acids and vectors for producing individual MAPP polypeptides and/or MAPP proteins (e.g., duplex MAPPs). The present disclosure also provides methods of producing MAPPs, duplex MAPPs, and higher order MAPPs utilizing such cells.
A. Definitions
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 “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids having a three-dimensional configuration, which unless stated otherwise are the naturally occurring proteinogenic L-amino acids that are incorporated biosynthetically into proteins during translation in a mammalian cell. Furthermore, as used herein, a “polypeptide” and “protein” include 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 to a specific residue or residue number in a known polypeptide, e.g., position 16 or 42 of human IL-2 polypeptide, are understood to refer to the amino acid at that position in the wild-type polypeptide (i.e. H16 or F42). To the extent that the sequence of the wild-type polypeptide is altered, either by addition or deletion of one or more amino acids, the specific residue or residue number will refer to the same specific amino acid in the altered polypeptide (e.g., in the addition of one amino acid at the N-terminus of a peptide reference as position H16, will be understood to indicate the amino acid, His, that is now position 16). Substitution of an amino acid at a specific position is denoted by an abbreviation comprising, in order, the original amino acid, the position number, and the substituted amino acid, e.g., substituting the His at position 16 with a Ala is denoted as H16A.
A nucleic acid or polypeptide has a certain percent “sequence identity” to another polynucleotide acid 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 blast.ncbi.nlm.nih.gov/Blast.cgi for BLAST+2.10.0, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, and mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Unless otherwise indicated, the percent sequence identities described herein are those determined using the BLAST program. In the event of a conflict between the results produced by different release versions of BLAST, BLAST+2.10.0 released Dec. 23, 2019, is employed as the basis for determining sequence identity.
As used herein amino acid (“aa” singular or “aas” plural) means the naturally occurring proteogenic amino acids incorporated into polypeptides and proteins in mammalian cell translation. Unless stated otherwise: L (Leu, leucine), A (Ala, alanine), G (Gly, glycine), S (Ser, serine), V (Val, valine), F (Phe, phenylalanine), Y (Tyr, tyrosine), H (His, histidine), R (Arg, arginine), N (Asn, asparagine), E (Glu, glutamic acid), D (Asp, asparagine), C (Cys, cysteine), Q (Gln, glutamine), I (Ile, isoleucine), M (Met, methionine), P (Pro, proline), T (Thr, threonine), K (Lys, lysine), and W (Trp, tryptophan) Amino acid also includes the amino acids hydroxyproline and selenocysteine, which appear in some proteins found in mammalian cells, however, unless their presence is expressly indicated they are not understood to be included.
As used herein the term “in vivo” refers to any process or procedure occurring inside of the body, e.g., of a patient.
As used herein, “in vitro” refers to any process or procedure occurring outside of the body.
The term “conservative amino acid substitution” refers to the interchangeability in proteins of aa residues having similar side chains. For example, a group of aas having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of aas having aliphatic-hydroxyl side chains consists of serine and threonine; a group of aas having amide containing side chains consists of asparagine and glutamine; a group of aas having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of aas having basic side chains consists of lysine, arginine, and histidine; a group of aas having acidic side chains consists of glutamate and aspartate; and a group of aas having sulfur containing side chains consists of cysteine and methionine. Exemplary conservative aa substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.
The term “binding” refers to a direct association between molecules and/or atoms, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
Non-covalent interactions/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. Non-covalent binding interactions are generally characterized by a dissociation constant (KD) of less than 10−6 M, less than 10−7 M, less than 10−8 M, less than 10−9 M, less than 10−10 M, less than 10−11 M, less than 10−12 M, less than 10−13 M, less than 10−14 M, or less than 10−15 M. “Covalent bonding,” or “covalent binding” as used herein, refers to the formation of one or more covalent chemical bonds between two different molecules. The term “binding,” as used with reference to the interaction between a MAPP and a T cell receptor (TCR) on a T cell, refers to a non-covalent interaction between the MAPP and TCR.
“Affinity” as used herein generally refers to the strength of non-covalent binding, increased binding affinity being correlated with a lower KD. As used herein, the term “affinity” may be described by the dissociation constant (KD) for the reversible binding of two agents (e.g., an antibody and an antigen. Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 40-fold greater, at least 60-fold greater, at least 80-fold greater, at least 100-fold greater, or at least 1,000-fold greater, or more, than the affinity of an antibody or receptor for an unrelated aa sequence (e.g., ligand). Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.
“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 as a “costimulatory polypeptide” or, as noted above, “MOD”), as used herein includes a wild-type or variant of a polypeptide or portion thereof that can specifically bind a cognate co-immunomodulatory polypeptide (“co-MOD”) present on a T cell, and provide a modulatory signal to the T cell when the TCR of the T cell is engaged with an MHC-epitope moiety that is specific for the TCR. Unless stated otherwise the term “MOD” includes wild-type and/or variant MODs, and statements including reference to both wild-type and variant MODs are made to emphasize that one, the other, or both are being referenced. The signal provided by the MOD engaging its co-MOD mediates (e.g., directs) a T cell response. Such responses include, but are not limited to, proliferation, activation, differentiation, suppression/inhibition of proliferation, activation and/or differentiation, and the like. A MOD can include, but is not limited to, 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, 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 also encompasses, inter alia, an antibody or antibody fragment that specifically binds with and activates a cognate co-stimulatory molecule (co-MOD) present on a T cell, such as, but not limited to antibodies against the receptors for any of IL-2, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-DC, B7-H2, B7-H3, and CD83.
“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.
The terms “treatment,” “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease or symptom in a mammal, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or symptom, i.e., arresting its development; and/or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, 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 humans and non-human primates, and in addition include rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), felines, canines, etc.
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 cell lysis” means an Ig Fc that induces no cell lysis at all or that largely but not wholly induces no cell lysis.
As used herein, the term “about” used in connection with an amount indicates that the amount can vary by 10%. 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.
The terms “purifying”, “isolating”, and the like, refer to the removal of a desired substance, e.g., a MAPP, from a solution containing undesired substances, e.g., contaminates, or the removal of undesired substances from a solution containing a desired substance, leaving behind essentially only the desired substance. In some instances, a purified substance may be essentially free of other substances, e.g., contaminates. Purifying, as used herein, may refer to a range of different resultant purities, e.g., wherein the purified substance makes up more than 80% of all the substance in the solution, including more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, more than 99.9%, and the like. As will be understood by those of skill in the art, generally, components of the solution itself, e.g., water or buffer, or salts are not considered when determining the purity of a substance.
Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range to a tenth of the lower limit of the range is encompassed within the disclosure along with any other stated or intervening value in the range. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, that are also encompassed within the disclosure subject to any specifically excluded limit in the stated range. Where the stated range a value (e.g., an upper or lower limit), ranges excluding those values are also included.
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.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a Treg” includes a plurality of such Tregs and reference to “the MHC Class I heavy chain” includes reference to one or more MHC Class I heavy chains 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.
B. Description
1. MAPP Structure and the Role of Framework and Dimerization Peptides
The present disclosure provides MAPPs for, among other things, use in the treatment of disease and disorders including cancers, autoimmune diseases, and infectious diseases. As discussed above, the MAPPs include at least one framework polypeptide and at least one dimerization polypeptide. Framework polypeptides comprise one or more polypeptide dimerization sequence that permits specific binding with other polypeptides (dimerization polypeptides) having a counterpart dimerization sequence thereby forming at least a heterodimer (see
As discussed above, the framework and dimerization peptide containing MAPPs, duplex MAPPs, and MAPPs of higher order (e.g., triplex MAPPs) described herein provide a means by which peptide epitopes may be delivered in the context of MHC (e.g., HLA) polypeptides to a target T cell displaying a TCR specific for the epitope, while at the same time permitting for the flexible presentation of one or more MODs. The MAPPs, duplex MAPPs, and higher order MAPPs thereby permit deliver of one or more MODs in an epitope selective (e.g., dependent/specific) manner that permits formation of an active immune synapse with a target T cell selective for the epitope, and control/regulation of the target T cell's response to the epitope. Accordingly, where MAPPs comprise stimulatory or activating MODs (e.g., IL-2, CD80, CD86, and/or 4-1BBL) that increase T cell proliferation and/or effector functions in an epitope selective manner In contrast, where MAPPs comprise suppressive/inhibitory MODs (e.g., FasL and/or PD-L1) they decrease T cell activation, proliferation, differentiation, and/or effector functions in an epitope selective manner
The framework/dimerization polypeptide architecture of MAPPs and their higher order structures may also be understood to provide flexibility in locating MODs and epitope presenting complexes or epitope presenting sequences. Duplex MAPP and higher order MAPP architecture can be particularly useful when both the MOD and the epitope presenting complexes (or epitope presenting sequences) are positioned so as to provide the desired biological activity as well as other desired properties of the MAPP, e.g., thermal stability and manufacturability. In some cases, acceptable combinations of properties may be obtained when the MOD and presenting complex or presenting sequence are positioned at the N-terminus of a polypeptide, e.g., each may be located at the N-terminus of different framework and/or dimerization polypeptide sequences. In some cases, acceptable combinations of properties may be obtained when the MOD and presenting complex or presenting sequence are positioned at the C-terminus of a polypeptide, e.g., each may be located at the C-terminus of different framework and/or dimerization polypeptide sequences. In some cases, acceptable combinations of properties may be obtained when the MOD and presenting complex or presenting sequence are positioned at the N-terminus and C-terminus of a polypeptide, respectively, e.g., the MOD may be located at the N-terminus and the presenting complex or presenting sequence may be located at the C-terminus of different framework and/or dimerization polypeptide sequences. In some cases, acceptable combinations of properties may be obtained when the MOD and presenting complex or presenting sequence are positioned at the C-terminus and N-terminus of a polypeptide, respectively, e.g., the MOD may be located at the C-terminus and the presenting complex or presenting sequence may be located at the N-terminus of different framework and/or dimerization polypeptide sequences.
The structure of MAPPs, and particularly higher order MAPPs such as duplexes, may be specified by the use of pairs of polypeptides having different sequences that specifically pair with each other. Multimerization of framework polypeptides results from interactions between multimerization sequences, and dimerization (the interaction of a framework and dimerization polypeptide) results from the interaction of a dimerization sequence on the framework polypeptide and a counterpart dimerization on a dimerization polypeptide. For example, in a duplex MAPP the multimerization sequences may be Ig Fc heavy chain (e.g., CH2-CH3) sequences, and the dimerization sequence and counterpart dimerization sequences may be the same (e.g., all leucine zipper sequences). An additional degree of control may be obtained by utilizing non-identical peptide sequences that specifically/selectively pair with each other that are referred to herein generally as “interspecific sequences,” in the case of dimerization sequences “interspecific dimerization sequences,” or in the case of multimerization sequences “interspecific multimerization sequences,” and which give rise to asymmetric interspecific pairs of sequences. The structure of MAPPs thus permits diverse and effective placement of each polypeptide into the MAPP architecture MAPP (see e.g.,
In an embodiment, the framework peptide multimerization sequence is an Fc heavy chain region (optionally a knob-in hole Fc sequence pair) and the dimerization sequences are the same (e.g., Ig CH1 sequences paired with light chain λ or κ constant region sequences) (see, for example,
MAPPs and accordingly their higher order complexes (duplexes, triplexes etc.) comprise MHC Class I polypeptide sequences that bind an epitope for presentation to a TCR, and accordingly may present peptides to T cells (e.g., CD8+ T cells). The effect of MAPPs on T cells with TCRs specific to the epitope depends on which, if any, MODs are present in the MAPP. As noted above, MAPPs, duplex MAPPS and higher order MAPPs comprising MOD(s) permit MOD delivery to T cells in an epitope selective manner and the MODs principally dictate the effect of MAPP-T cell engagement in light of the specific cell type stimulated and the environment. While not wishing to be bound by any particular theory, the effect of MAPP (e.g., duplex MAPP) presentation of MOD(s) and epitope to a T cells in some cases may be enhanced relative to the situation encountered in antigen presenting cells (APC) where epitope can diffuse away from the MHC-H/β2M complex and any MODs the APC is presenting. This situation may not occur where the epitope and MOD(s) are part of the MAPP polypeptide(s) and cannot diffuse away even if the epitope's affinity for the MHC-H/β2M complex would normally permit it to leave the comparable cell complex. The inability of epitope to diffuse away from MHC-H/β2M and MOD components of a MAPP, duplex MAPP, or higher order MAPP may be further limited where the polypeptide(s) of the MAPP (e.g., framework, dimerization sequence, and if present, the presenting complex 2nd sequence) are covalently attached to each other (e.g., by disulfide bonds). Consequently, MAPPs and their higher order structures can prolong delivery of MOD(s) to T cells in an epitope selective manner relative to systems where epitope can diffuse away from the presenting MHC. In the absence of any MOD or any stimulatory MOD, prolonged exposure to the MAPP may result in T cell anergy or suppression of T cell stimulation.
Incorporation of one or more MODs with affinity for their cognate receptor on T cells (“co-MOD”) can reduce the specificity of MAPPs (e.g., duplex MAPPs) for epitope selective/specific T cells. The reduction in epitope selectivity/specificity of the MAPPs becomes more pronounced where MOD/co-MOD binding interactions increase in strength (binding energy) and significantly compete with MHC/epitope binding to target cell TCR. The inclusion of variant MODs with reduced affinity for their co-MOD(s) thus may provide a lower contribution of MOD binding energy, thereby permitting MHC-epitope interactions in which the TCR dominates the binding and provides epitope selective interactions with T cells while retaining the activity of the MODs. Variant MODs with one or more substitutions (or deletions or insertions) that reduced the affinity of the MOD for their co-MOD may be incorporated into MAPPs and their higher order complexes alone or in combination with wild-type MODs polypeptide sequences. Wild-type and variant MODs are described further below. The ability of MAPPs to modulate T cells in an epitope selective/specific manner thus provides methods of modulating activity of a T cell in vitro and in vivo, and accordingly, methods of treating disease such as cancers, infections, and disorders related to immune dysregulation/disfunction, including allergies and autoimmune diseases.
The present disclosure provides nucleic acids comprising nucleotide sequences encoding MAPP polypeptides, cells genetically modified with the nucleic acids and capable of producing the MAPP, and methods of producing MAPPs and their higher order complexes utilizing such cells.
Each presenting sequence or presenting complex present in a MAPP comprises MHC Class I heavy chain and β2M polypeptide sequences (e.g., human MHC Class I sequences) sufficient to bind a peptide epitope and present it to a TCR. MHC Class I peptides, may include sequence variations that are designed to stabilize the MHC, stabilize the MHC peptide epitope complex, and/or stabilize the MAPP. Sequence variations may also serve to enhance cellular expression of MAPPs prepared in cell-based systems as well as the stability (e.g., thermal stability) of MAPPs and their higher order complexes such as duplex MAPPs. Some MHC Class I sequences suitable for use in MAPPs are described below.
As indicated in the description of the drawings, MAPPs may comprise one or more independently selected peptide sequences or (one or more “linker” or “linkers”) between any two or more components of the MAPP, which in the figures may be shown as a line between peptide and/or polypeptide elements of the MAPPs. The same sequences used as linkers may also be located at the N- and/or C-termini of the MAPP peptides to prevent, for example, proteolytic degradation. Linker sequences include but are not limited to polypeptides comprising: glycine; glycine and serine; glycine and alanine; alanine and serine; and glycine, alanine and serine; any one which may comprise a cysteine for formation of an intra or interpeptide disulfide bond. Various linkers are described in more detail below.
MAPPs of the present disclosure comprise (i)framework polypeptides with a multimerization sequence and at least one dimerization sequence, and (ii) dimerization polypeptides with a counterpart dimerization sequence that binds with the framework polypeptide's the dimerization sequence. As discussed above, MAPPs typically will further comprise either one or more epitope presenting sequences or one or more epitope presenting complexes. Exemplary structures for such MAPPs appear in
Interactions of MHC-H and β2M are not considered to result in multimerization and/or dimerization. In and embodiment, neither the dimerization sequence nor the multimerization sequence of the framework polypeptide, nor the counterpart dimerization sequence of the dimerization polypeptide comprises a Class I MHC-H or β2M polypeptide sequence having at least 90% (e.g., 95% or 98%) sequence identity to at least 15 (e.g., at least 20, 30, 40, 50, 60 or 70) contiguous aas of a MHC Class I polypeptide(e.g., a polypeptide in any of
One group of MAPPs, those having epitope presenting sequences, comprise a multimerizing framework polypeptide having, from N-terminus to C-terminus, a dimerization sequence and multimerization sequence; and a dimerization polypeptide comprising a counterpart dimerization sequence complementary to the dimerization sequence of the framework polypeptide and dimerizing therewith through covalent and/or non-covalent interactions to form a heterodimer; wherein at least one (e.g., one, or both) of a dimerization polypeptide and the framework polypeptide comprise a presenting sequence located on the N-terminal side of their dimerization or counterpart dimerization sequences. In such a MAPP the presenting sequence may comprise a peptide epitope and one or more MHC polypeptide sequences, with the peptide epitope sequence located: (i) at or within 10 aa, 15 aa, 20 aa, or 25 aa of the N-terminus of the presenting sequence, or (ii) in a polypeptide located at the N-terminus of the presenting sequence comprising, from N-terminus to C-terminus, a MOD, one or more optional linkers, and the peptide epitope; optionally at least one (e.g., one, two or each) of the framework polypeptide, dimerization peptide, and presenting sequence comprises one or more independently selected MODs located at their N-terminus and/or C-terminus (or on the N-terminal or C-terminal side of the dimerization or counterpart dimerization sequences); wherein the MHC polypeptide sequences are MHC Class I polypeptide sequences and they comprise an MHC Class I heavy chain (e.g., MHC-H α1, α2, and α3 domains) polypeptide and β2M polypeptide sequences (e.g., human MHC-H and a β2M sequences). In an embodiment, neither the dimerization sequence nor the multimerization sequence of the framework polypeptide comprises a MHC-H peptide or β2M sequence having at least 90% (e.g., 95% or 98%) sequence identity to at least 15 (e.g., at least 20, 30, 40, 50, 60 or 70) contiguous aas of a MHC Class I polypeptide in any of
Another group of MAPPs, those having epitope presenting complexes, comprise a framework polypeptide having, from N-terminus to C-terminus, a dimerization sequence and multimerization sequence; and a dimerization polypeptide comprising a counterpart dimerization sequence complementary to the dimerization sequence of the framework polypeptide and dimerizing therewith through covalent and/or non-covalent interactions to form a heterodimer; wherein at least one (e.g., one, or both) of a dimerization polypeptides and/or at least one (e.g., one or both) of the framework polypeptide comprise a presenting complex 1st sequence located on the N-terminal side of their dimerization sequence. A presenting complex 2nd sequence is associated with the presenting complex 1st sequence (e.g., non-covalently or covalently such as by one or two interchain disulfide bonds) to form a presenting complex. In such a MAPP, each of the presenting complex 1st sequence and its associated presenting complex 2nd sequence are comprised of one or more the MHC-H and β2M polypeptide sequences, with one of the sequences further comprising the peptide epitope. The peptide epitope may be located (i) at or within 10aa, 15 aa, 20 aa, or 25 aa of the N-terminus of the presenting complex 1st sequence or presenting complex 2nd sequence, or (ii) in a polypeptide located at the N-terminus of the presenting complex 1st sequence or presenting complex 2nd sequence, with the polypeptide comprising, from N-terminus to C-terminus, a MOD, one or more optional linkers, and the peptide epitope. Optionally, at least one (e.g., one, two or each) of the framework polypeptide, dimerization peptide, or the peptides of a presenting complex comprise one or more independently selected MODs located at their N-terminus or C-terminus (or on the N-terminal or C-terminal side of the dimerization sequences).
MAPPs of the present disclosure may be constructed such that neither the dimerization sequence nor the multimerization sequence of the framework polypeptide comprises a Class I MHC peptide sequence having at least 90% (e.g., 95% or 98%) sequence identity to at least 15 (e.g., at least 20, 30, 40, 50, 60 or 70) contiguous aas of a MHC Class I polypeptide in any of
As discussed above, a dimerization sequence of a framework polypeptide may interact with dimerization peptides to form heterodimers. The multimerization sequence of the framework polypeptide may associate with another framework polypeptide multimerization sequence forming a duplex (or higher order structure, such as a triplex, quadraplex or pentaplex) of the heterodimers. Where the multimerization sequences are interspecific (e.g., a knob-in-hole Fc peptide pair), and at least one heterodimer comprises an interspecific dimerization and counterpart dimerization pair, two different heterodimers may be formed. When the different heterodimers are combined to form a duplex MAPP, any one or more component (e.g., MODs) may differ (e.g., in type or location) between the two heterodimers.
C. MAPP Components 1. Framework Polypeptides and Dimerization Polypeptides
As may be understood from the preceding sections, framework polypeptides serve as the structural basis or skeleton of MAPPs, permitting the organization of other elements in the MAPP complex. Framework peptides interact with other peptides through binding interactions, principally at dimerization and multimerization sequences. Interactions at dimerization sequences permit association of non-framework peptides (e.g., dimerization peptides) with framework peptides. In contrast, multimerization sequences are involved in the interaction of two or more framework peptides.
The framework polypeptide(s) of MAPPs comprise at least one multimerization sequence, and at least one independently selected dimerization sequence that is not identical to or of the same type (e.g., not both leucine zipper variants) as the multimerization sequence. By utilizing different types of sequences for the interactions at multimerization and dimerization sequences, it becomes possible to control the interactions of the framework polypeptide with other framework polypeptides and with dimerization polypeptides. In an embodiment, framework polypeptides comprise one multimerization sequence and one dimerization sequence. In an embodiment, framework polypeptides comprise at least one multimerization sequence and at least two independently selected dimerization sequences. Framework peptides may contain peptide sequences (e.g., linker sequences and/or MOD sequences) between any of the elements of the framework polypeptide or at the ends of the framework polypeptide including the multimerization sequences and dimerization sequences.
In addition to providing for the structural organization of MAPPS through their multimerization and dimerization sequences, framework peptides, and particularly their N- and C-termini, may also serve as locations for placement of elements such as MOD sequences, an epitope, presenting sequences, and/or a presenting complex 1st sequence (one polypeptide of an epitope presenting complexes), see
Within a MAPP, all of the dimerization sequence may be non-interspecific (such as leucine zipper pairs) while the multimerization sequences is either interspecific or non-interspecific (see e.g., structures A & B of
Within a MAPP, all of the dimerization sequences may be interspecific, while the multimerization sequences are not interspecific (see e.g.,
All of the dimerization sequences or all of the dimerization and multimerization sequences, in a MAPP may differ in that they bind only specific binding partners present in the MAPP (e.g., each are part of a different interspecific sequence pair). For example, in a duplex MAPP with first and second framework polypeptides, the multimerization sequences may be a pair of knob-in-hole IgFc sequences, with the a ZW1 sequence or its counterpart employed as the dimerization sequence of the first framework polypeptide, and an Ig CH1 or its counterpart Ig CL sequence as the dimerization sequence of the second framework polypeptide.
Amino acid sequences that permit polypeptides to interact may be utilized as dimerization sequences or counterpart dimerization sequences when they are involved in the formation of dimers between a framework polypeptide and a dimerization polypeptide. The same type of aa sequences may be utilized as multimerization sequences when they are used to form duplex or higher order structures (trimers, tetramers, pentamer, etc.) between framework polypeptides. In any given MAPP, sequences that can interact with each other are not utilized as both dimerization and multimerization sequences. Stated another way, the same aa sequence pair may serve as either dimerization or multimerization sequences depending on whether they: bring together two or more framework peptides, in which case they are multimerization sequences; or they bring together a dimerization and multimerization sequence, in which case they are designated as dimerization sequences.
Where dimerization or multimerization sequences employ identical sequences that pair or multimerize (e.g., some leucine zipper sequences), they can form symmetrical pairs or multimers (e.g., homodimers) as shown in
Interspecific binding sequences may in some instances form some amount of homodimers, but preferentially dimerize by binding more strongly with their counterpart interspecific binding sequence. Accordingly, specific heterodimers tend to be formed when an interspecific dimerization sequence and its counterpart interspecific binding sequence are incorporated into a pair of polypeptides. By way of example, where an interspecific dimerization sequence and its counterpart are incorporated into a pair of polypeptides, they may selectively form greater than 70%, 80%, 90%, 95%, 98% or 99% heterodimers when an equimolar mixture of the polypeptides are combined (for example in PBS buffer at 20° C.). The remainder of the polypeptides may be present as monomers or homodimers, which may be separated from the heterodimer. See, for example,
Sequence are considered orthogonal to other sequences when they do not form complexes (bind) with each other's counterpart sequences. See
Some sequences permitting polypeptides to interact with sufficient affinity to be used as dimerization and/or multimerization sequences are provided for example in U.S. Patent Publication No. 2003/0138440. The sequences may be of relatively compact size (e.g., such as less than about 300, 250, 225, 200, 175, 150, 125, 100, 75, 60, 50, 40, or 30 aa). In an embodiment, at least one (e.g., at least two or all) dimerization and/or multimerization sequence is less than 300 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and/or multimerization sequences are less than 200 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and/or multimerization sequences are less than 100 aa. In an embodiment, at least one (e.g., at least two or all) or all dimerization and/or multimerization sequences are less than 75 aa. In another embodiment, at least one (e.g., at least two or all) of the dimerization and/or multimerization sequences are less than are less than 50 aa. In an embodiment, at least one (e.g., at least two or all) of the dimerization and/or multimerization sequences are less than 30 aa.
Dimerization/multimerization sequences include but are not limited to immunoglobulin heavy chain constant region (Ig Fc) polypeptide sequences (e.g., sequences comprising CH2-CH3 regions of immunoglobulins such as those provided in
Framework and/or dimerization polypeptides of a MAPP may comprise an immunoglobulin heavy chain constant region (e.g., CH2-CH3 domains) polypeptide sequence that functions as a dimerization or multimerization sequence. Where the framework polypeptide comprises a IgFc multimerization sequence, and a CH1 dimerization sequence it may comprise all or part a native or variant immunoglobin sequence set forth in any of
Such immunoglobulin sequences can covalently link the polypeptides of MAPP complex together by forming one or two interchain disulfide bonds, thereby stabilizing MAPPs, particularly where a pair of interspecific Ig sequence such as knob-in-hole polypeptide pairs are employed. Where an Fc polypeptide sequence, alone or in combination with a CH1 polypeptide sequence, is employed as a multimerization or dimerization sequence it may be, for example, from an IgA, IgD, IgE, IgG, or IgM, which may be a human polypeptide sequence, a humanized polypeptide sequence, a Fc region polypeptide of a synthetic heavy chain constant region, or a consensus heavy chain constant region. As discussed below, the Ig Fc region can further contain substitutions that can substantially reduce the ability of the Ig Fc to effect complement-dependent cytotoxicity (CDC) or antibody-dependent cell cytotoxicity (ADCC). Accordingly, framework and/or dimerization polypeptides, and in particular Ig Fc sequences used as multimerization or dimerization sequences, may comprise substitutions that reduce or substantially eliminate ADCC and/or CDC responses.
Framework and/or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 150 contiguous aas (at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, or at least 350 contiguous aas), or all aas, of the IgA Fc sequence depicted in
A MAPP may comprise one or more IgG Fc sequences as dimerization and/or multimerization sequences. The Fc polypeptide of a MAPP of the present disclosure can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, etc. In some cases, the Fc sequence has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to an aa sequence of an Fc region depicted in
Framework and/or dimerization polypeptides of a MAPP may comprise a sequence that has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to at least 125 (at least 150, at least 175, at least 200, at least 225, or at least 250) contiguous aas, or all aas, of the IgM Fc polypeptide sequence depicted in
Framework and/or dimerization polypeptides of a MAPP comprising immunoglobulin sequences (e.g., depicted in
A framework or dimerization polypeptide may comprise an aa sequence having 100% aa sequence identity to the wt. human IgG1 Fc polypeptide depicted in
Amino acid L234 and other aas in the lower hinge region (e.g., aas 234 to 239, such as L235, G236, G237, P238, S239), which correspond to aas 14-19 of SEQ ID NO:57) of IgG are involved in binding to the Fc gamma receptor (FcγR), and accordingly, mutations at that location reduce binding to the receptor (relative to the wt. protein) and result in a reduction in antibody-dependent cellular cytotoxicity (or alternatively antibody-dependent cell-mediated cytotoxicity, “ADCC”). Hezareh et al., (2001) have demonstrated that the double mutant (L234A, L235A) does not effectively bind either FcγR or C1q, and both ADCC and CDC functions were reduced or substantially eliminated. A framework or dimerization polypeptide with a substitution in the lower hinge region may comprise an aa sequence having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to at least 125 contiguous aas (e.g., at least 150, at least 175, at least 200, or at least 210 contiguous aas), or all aas, of the wt. human IgG1 Fc polypeptide depicted in
A framework or dimerization polypeptide with a substitution in the lower hinge region may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 Fc polypeptide depicted in
A framework or dimerization polypeptide with a substitution in the lower hinge region may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 Fc polypeptide depicted in
A framework or dimerization polypeptide may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 Fc polypeptide depicted in
A framework or dimerization polypeptide may comprise an aa sequence (e.g., as a multimerization sequence) having at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, or 99%) aa sequence identity to the wt. human IgG1 Fc polypeptide depicted in
Where an asymmetric pairing between two polypeptides of a MAPP is desired, a framework or dimerization polypeptide present in a MAPP may comprise, consist essentially of, or consist of an interspecific binding sequence. 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 immunoglobulin Fc (Ig Fc) sequence variants. Such interspecific polypeptide sequences include knob-in-hole without (KiH) or with (KiHs-s) a stabilizing disulfide bond, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequences. 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 Ig 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). Several interspecific binding sequences based upon immunoglobulin sequences are summarized in the table that follows, with cross reference to the numbering of the aa positions as they appear in the wt. IgG1 sequence (SEQ ID NO:57) set forth in
In addition to the interspecific pairs of sequences in Table 1, framework and/or dimerization polypeptides may include interspecific “SEED” sequences having 45 residues derived from IgA in an IgG1 CH3 domain of the interspecific sequence, and 57 residues derived from IgG1 in the IgA CH3 in its counterpart interspecific sequence. See Ha et al., Frontiers in Immunol.7:1-16 (2016).
A framework or dimerization polypeptide found in a MAPP may comprise an interspecific binding sequence or its counterpart interspecific binding sequence selected from the group consisting of: KiH); (KiHs-s); HA-TF; ZW-1; 7.8.60; DD-KK; EW-RVT; EW-RVTs-s; A107; or SEED sequences.
A MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 KiH or KiHs-s sequence with a T146W sequence substitution, and its counterpart interspecific KiH or KiHs-s binding partner polypeptide comprises an IgG1 sequence having T146S, L148A, and Y187V sequence substitutions, where the framework and/or dimerization polypeptides comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
A MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 sequence with a T146W KiH sequence substitution, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T146S, L148A, and Y187V KiH sequence substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
A MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 sequence with a T146W and S134C KiHs-s substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T146S, L148A, Y187V and Y129C KiHs-s substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
A MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 sequence with a S144H and F185A HA-TF substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having Y129T and T174F HA-TF substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
A MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 sequence with a T130V, L131Y, F185A, and Y187V ZW1 substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V, T146L, K172L, and T174W ZW1 substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
A MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 sequence with a K140D, D179M, and Y187A 7.8.60 substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V E125R, Q127R, T146V, and K189V 7.8.60 substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
A MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 sequence with a K189D, and K172D DD-KK substitutions, and its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V D179K and E136K DD-KK substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
A MAPP may comprise a framework or dimerization polypeptide comprising an IgG1 sequence with a K140E and K189W EW-RVT substitutions, its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V Q127R, D179V, and F185T EW-RVT substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 sequence with a K140E, K189W, and Y129C EW-RVTs-s substitutions, its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V Q127R, D179V, F185T, and S134C EW-RVTs-s substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
In an embodiment, a MAPP comprises a framework or dimerization polypeptide comprising an IgG1 sequence with a K150E and K189W A107 substitutions, its counterpart interspecific binding partner polypeptide comprises an IgG1 sequence having T130V E137N, D179V, and F185T A107 substitutions, where the framework and/or dimerization polypeptides comprise a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity to at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous aas of the wt. IgG1 of
As an alternative to the use of immunoglobulin CH2 and CH3 heavy chain constant regions as dimerization or multimerization sequences, immunoglobulin light chain constant regions (See
In an embodiment, a MAPP framework or dimerization polypeptide comprises an Ig CH1 domain (e.g., the polypeptide of
A framework or dimerization polypeptide of a MAPP comprises an Ig CH1 domain (e.g., the polypeptide of
Framework and/or dimerization polypeptides of a MAPP each comprise a leucine zipper polypeptide as a dimerization or multimerization sequence. The leucine zipper polypeptides bind to one another to form dimer (e.g., homodimer). Non-limiting examples of leucine-zipper polypeptides include a peptide comprising any one of the following aa sequences: RMKQIEDKIEEILS-KIYHIENEIARIKKLIGER (SEQ ID NO:71); LSSIEKKQEEQTSWLIWISNELTLIRNELAQS (SEQ ID NO:72); LSSIEKKLEEITSQLIQISNELTLIRNELAQ (SEQ ID NO:73; LSSIEKKLE-EITSQLIQIRNELTLIRNELAQ (SEQ ID NO:74); LSSIEKKLEEITSQLQQIRNELTLIRNELAQ (SEQ ID NO:75); LSSLEKKLEELTSQLIQLRNELTLLRNELAQ (SEQ ID NO:76); ISSLEKKIEELTSQI-QQLRNEITLLRNEIAQ (SEQ ID NO:77). In some cases, a leucine zipper polypeptide comprises the following aa sequence: LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK (SEQ ID NO:78). Additional leucine-zipper polypeptides are known in the art, a number of which are suitable for use as multimerization or dimerization sequences.
The framework and/or dimerization polypeptides of a MAPP may comprise a coiled-coil polypeptide sequence that forms a dimer. Non-limiting examples of coiled-coil polypeptides include, for example, a peptide of any one of the following aa sequences: LKSVENRLAVVENQLKTVIEELK-TVKDLLSN (SEQ ID NO:79); LARIEEKLKTIKAQLSEIASTLNMIREQLAQ (SEQ ID NO:410); VSRLEEKVKTLKSQVTELASTVSLLREQVAQ (SEQ ID NO:80); IQSEKKIEDISSLIG-QIQSEITLIRNEIAQ (SEQ ID NO:81); and LMSLEKKLEELTQTLMQLQNELSMLKNELAQ (SEQ ID NO:82).
A MAPP may comprise a pair of two framework polypeptides and/or a framework and dimerization polypeptide that each have an aa sequence comprising at least one cysteine residue that can form a disulfide bond permitting homodimerization or heterodimerization of those polypeptides stabilized by disulfide bond between the cysteine residues. Examples of such aa sequences include:
Some aa sequences suitable as multimerization (oligomerization) sequences permit formation of MAPPs capable of forming structures greater than duplexes of a heterodimers comprising a framework and dimerization polypeptide. In some instances, triplexes, tetraplexes, pentaplexes may be formed. Such aa sequences include, but are not limited to, IgM constant regions (see e.g.,
Suitable framework polypeptides (e.g., those with an Ig Fc multimerization sequence) will, in some cases, be half-life extending polypeptides. Thus, in some cases, a suitable framework polypeptide increases the in vivo half-life (e.g., the serum half-life) of the MAPPs, compared to a control MAPP having a framework polypeptide with a different aa sequence. For example, in some cases, a framework polypeptide increases the in vivo half-life (e.g., the serum half-life in a mammal such as a human) of the MAPP, compared to a control MAPP having a framework polypeptide with a different aa sequence. The half-life may be extended by at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 100%, at least about 2-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 Ig Fc polypeptide sequence (e.g., utilized as a multimerization sequence to form a duplex of MAPP heterodimers comprising a framework and dimerization polypeptide) increases the stability and/or in vivo half-life (e.g., the serum half-life) of a MAPP duplex compared to a control MAPP duplex lacking the Ig Fc polypeptide sequence by at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 100%, 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.
Presenting sequences (epitope presenting sequences) and presenting complexes (epitope presenting complexes) comprise MHC Class I polypeptides (MHC-H and β2M) sufficient to bind and present an epitope to a TCR. Presenting sequences and complexes may also comprise additional protein (peptide) elements including one or more independently selected MODs and/or one or more independently selected linkers (e.g., linkers placed between various domains). As discussed herein, unless stated otherwise, neither presenting sequences nor presenting complexes comprise an MHC transmembrane domain (or intracellular domain such as a cytoplasmic tail) sufficient to anchor MAPP molecules (e.g., more than 50% of the MAPP molecules) in a mammalian cell membrane (e.g., a CHO cell membrane) when expressed therein.
Conceptually, each of the presenting sequences and presenting complexes may be considered a “soluble MHC” that is fully capable of binding and presenting a peptide epitope (e.g., and peptide epitope that is part of a polypeptide comprising an MHC sequence) to a TCR. In presenting sequences all of the MHC sequences and optionally the peptide epitope sequence are present in a single polypeptide chain (single linear sequence of aas produced by translation). See e.g.,
Where the MHC-H and β2M polypeptide sequences are divided among two or more polypeptide chains the “soluble MHC” is termed a presenting complex. The presenting complex has one chain that is part of a framework peptide or dimerization peptide, referred to as a “presenting complex 1st sequence.” The second chain of the presenting complex is termed the “presenting complex 2nd sequence.” The presenting complex 2nd sequence may be associated non-covalently with the MHC components present in the presenting complex 1st sequence (through binding interactions between MHC-H and β2M components as in
In some cases, one or more presenting sequence of a MAPP comprises all of the Class I components required for binding and presenting the epitope of interest to a TCR; e.g., the MHC-H and β2M and epitope in a single polypeptide sequence. In some cases, a MAPP comprises one or more presenting complexes with all of the Class I components required for binding and presenting the epitope of interest to a TCR, where the epitope is part of the presenting complex 1st sequence or the presenting complex 2nd sequence.
Peptide presenting sequences and peptide presenting complexes in MAPPs may contain one or more substitutions in, for example, the MOD sequences and/or MHC sequences. By way of example, the presenting proteins or complexes may contain one or more reduced affinity variant MODs. The presenting sequences or complexes may also comprise substitutions in their MHC sequences, such as a Class I MHC containing a Y84A substitution (as discussed above, that opens the end of the binding pocket). A Class I MHC containing presenting sequence or presenting complex may also contain substitutions leading to stabilizing disulfide bonds, such as a cysteine substituted into the carboxyl end portion of the al helix and a cysteine in the amino end portion of the α2-1 helix (e.g., a disulfide bond formed between positions 84 and 139 in a Y84C and A139C substituted MHC-H as previously discussed). A Class I MHC containing presenting sequence or presenting complex bearing cysteine substitutions at MHC-H aa 236 (A236C) and β2M aa 12 (e.g., R12C) may form a disulfide bond between those aas. In presenting sequences or presenting complexes having peptide epitopes bound by a linker to the N-terminal side of the β2M sequence(s), a stabilizing disulfide bond joining a cysteine residue present in the linker (e.g., at position 2 of a the linker such as a G2C substitutions in the linkers of SEQ ID NOs:131-133 and 571 provided below) and a cysteine residue in the MHC-H polypeptide (e.g., a Cys introduced at position 84 such as a Y84C substitution) may also be introduced. A Class I MHC containing presenting sequence or presenting complex may also contain paired substitutions forming two disulfide bonds such as between the R12C/Y84C and the Y84C/linker (e.g., G2C) substitution pairs.
For the purpose of this disclosure, the term “MHC polypeptide” is meant to include Class I MHC polypeptides, including MHC-H polypeptide sequences (e.g., α1, α2, and α3 MHC-H polypeptide domains) and the β-2 microglobulin (β2M) polypeptide sequences, which represent all or most of the extracellular Class I protein required for presentation of an epitope to a TCR. Accordingly, the MHC Class I heavy chain present in a MAPP of this disclosure may include only MHC Class I heavy chain α1, α2, and α3 domains. In some cases, the MHC Class I heavy chain sequence present in a MAPP of the present disclosure has a length from about 270 aa to about 290 aa. In some cases, the MHC Class I heavy chain present in a MAPP of the present disclosure has a length of 270 aa, 271 aa, 272 aa, 273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa, 280 aa, 281 aa, 282 aa, 283 aa, 284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290 aa.
The epitope-containing MAPPs include MHC polypeptides of various species, including human MHC polypeptides (HLA polypeptides), rodents (e.g., mouse, rat, etc.), and the 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. Both the β2M and MHC-H chain sequences in a MAPP may be of human origin.
MAPPs and their higher order complexes (e.g., duplex MAPPs) are intended to be soluble in aqueous media under physiological conditions (e.g., soluble in human blood plasma at therapeutic levels). Unless expressly stated otherwise, as noted above, the MAPPs described herein are not intended to include membrane anchoring domains (such as transmembrane regions of MHC Class I polypeptides, or a part thereof sufficient to anchor MAPP molecules (e.g., more than 50% of the MAPP molecules), or a peptide thereof, in the membrane of a cell (e.g., a eukaryotic cell such as a mammalian cell such as a Chinese Hamster Ovary or “CHO” cell) in which the MAPP is expressed. Similarly, unless expressly stated otherwise, the MAPPs described herein do not include the leader and/or intracellular portions (e.g., cytoplasmic tails) that may be present in some naturally-occurring MHC Class I proteins
In some cases, a MHC polypeptide of a MAPP is a human MHC polypeptide, and may be referred to as “human leukocyte antigen” (“HLA”) polypeptides, more specifically, a Class I HLA polypeptide, e.g., a β2M polypeptide, or a Class I HLA heavy chain polypeptide. Class I HLA heavy chain polypeptides that can be included in MAPPs include, for example, HLA-A, -B, -C, -E, -F, and/or -G heavy chain polypeptide extracellular domains (e.g., fully process and lacking the signal sequence). In an embodiment, the Class I HLA heavy chain polypeptides of MAPPs comprise polypeptides having a 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% aa sequence identity to all or part (e.g., 50, 75, 100, 150, 200, or 250 contiguous aa) of the aa sequence of any of the human HLA heavy chain polypeptides depicted in
As an example, a MHC Class I heavy chain polypeptide of a MAPP can comprise an aa 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% aa sequence identity to aas 25-300 (lacking all, or substantially all, of the leader, transmembrane and cytoplasmic sequences), or aas 25-365 (lacking the leader), of the human HLA-A heavy chain polypeptides depicted in
An HLA heavy chain polypeptides (human MHC-H) of a MAPP may comprise a polypeptide sequence having at least 95%, sequence identity (e.g., 95-100%) sequence identity to all or part (e.g., 50, 75, 100, 150, 200, or 250 contiguous aa) of any one of the HLA heavy chain polypeptides in
An HLA heavy chain polypeptides of a MAPP may comprise a polypeptide sequence having at least 95%, sequence identity (e.g., 95-100%) sequence identity to at least 125 contiguous aa of any one of the HLA heavy chain polypeptides in
HLA heavy chain polypeptides of a MAPP may comprise a polypeptide sequence having at least 95%, sequence identity (e.g., 95-100%) sequence identity to at least 175 contiguous aa of any one of the HLA heavy chain polypeptides in
HLA heavy chain polypeptides of a MAPP may comprise a polypeptide sequence having at least 95%, sequence identity (e.g., 95-100%) sequence identity to at least 225 contiguous aa of any one of the HLA heavy chain polypeptides in
HLA heavy chain polypeptides of a MAPP may comprise a polypeptide sequence having at least 95%, sequence identity (e.g., 95-100%) sequence identity to at least 250 contiguous aa of any one of the HLA heavy chain polypeptides in
(i) MHC Class I Heavy Chains
Class I human MHC polypeptides may be drawn from the classical HLA alleles (HLA-A, B, and C), or the non-classical HLA alleles (e.g., HLA-E, F and G). The following are non-limiting examples of MHC-H alleles and variants of those alleles that may be incorporated into MAPPs.
(a) HLA-A Heavy Chains
The HLA-A heavy chain peptide sequences, or portions thereof, that may be incorporated into a MAPP include, but are not limited to, the alleles: A*0101 (
HLA-A*0101 (HLA-A*01:01:01:01)
In an embodiment, a MHC Class I heavy chain polypeptide of a MAPP comprises an aa sequence of HLA-A*01:01:01:01 (SEQ ID NOs: 12 or 23), or a 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%) aa sequence identity to all or part (e.g., 50, 75, 100, 150, 200, or 250 contiguous aas) of that sequence (e.g., it may comprise 1-30, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 aa insertions, deletions, and/or substitutions). In an embodiment, where the HLA-A heavy chain polypeptide of a MAPP has less than 100% identity to the sequence labeled HLA-A in
HLA-A*0201 (HLA-A*02:01)
In an embodiment, a MHC Class I heavy chain polypeptide of a MAPP comprises an aa sequence of HLA-A*0201 (SEQ ID NOs: 15 or 24) provided in
HLA-A*1101 (HLA-A*11:01)
In an embodiment, a MHC Class I heavy chain polypeptide of a MAPP comprises an aa sequence of HLA-A*1101(SEQ ID NOs: 20 or 26) provided in
In an embodiment, where the HLA-A*1101 heavy chain polypeptide of a MAPP has less than 100% identity to the sequence labeled HLA-A*1101 in
HLA-A*2402 (HLA-A*24:02)
In an embodiment, a MHC Class I heavy chain polypeptide of a MAPP comprises an aa sequence of HLA-A*2402 (SEQ ID NOs: 21 or 28) provided in
In an embodiment, where the HLA-A*2402 heavy chain polypeptide of a MAPP has less than 100% identity to the sequence labeled HLA-A*2402 in
HLA-A*3303 (HLA-A*33:03)
In an embodiment, a MHC Class I heavy chain polypeptide of a MAPP comprises an aa sequence of HLA-A*3303 (SEQ ID NOs: 22 or 30) provided in
In an embodiment, where the HLA-A*3303 heavy chain polypeptide of a MAPP has less than 100% identity to the sequence labeled HLA-A*3303 in
(b) HLA-B Heavy Chains
The HLA-B heavy chain peptide sequences, or portions thereof, that may be incorporated into a MAPP include, but are not limited to, the alleles B*0702 shown in
HLA-B*0702 (HLA-B*07:02)
In an embodiment, a MHC Class I heavy chain polypeptide of a MAPP comprises an aa sequence of HLA-B*0702 (SEQ ID NO:33 and SEQ ID NO:13 in
(c) HLA-C Heavy Chains
The HLA-C heavy chain peptide sequences, or portions thereof, that may be incorporated into a MAPP include, but are not limited to, the alleles: C*0102 (SEQ ID NO:41), C*0303 (SEQ ID NO: 42), C*0304 (SEQ ID NO:43), C*0401 (SEQ ID NO:44), C*0602 (SEQ ID NO:45), C*0701(SEQ ID NO:46), C*0702 (SEQ ID NO:47), C*0801 (SEQ ID NO:48), and C*1502 (SEQ ID NO:49), which are aligned without all, or substantially all, of the leader, transmembrane and cytoplasmic sequences in
HLA-C*701 (HLA-C*07:01) and HLA-C*702 (HLA-C*07:02)
The MHC Class I heavy chain polypeptide of a MAPP may comprise an aa sequence of HLA-C*701 (
(d) Non-Classical HLA-E, F and G Heavy Chains
The non-classical HLA heavy chain peptide sequences, or portions thereof, that may be incorporated into a MAPP include, but are not limited to, those of the HLA-E, F, and/or G alleles. Sequences for those alleles (and the HLA-A, B and C alleles) may be found on the world wide web at, for example, hla.alleles.org/nomenclature/index.html, the European Bioinformatics Institute (www.ebi.ac.uk), which is part of the European Molecular Biology Laboratory (EMBL), and at the National Center for Bioecology Information (www.ncbi.nlm.nih.gov).
Some 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. Some 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. Some 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*01:11, 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. 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
(e) Mouse H2K
In an embodiment, a MHC Class I heavy chain polypeptide of a MAPP comprises an aa sequence of MOUSE H2K (SEQ ID NO:16) (MOUSE H2K in
(f) The Effect of Substitutions and Combinations of Substitutions in Class I Polypeptides on MAPPS
Substitution of position 84, particularly when it is a tyrosine residue, with a small aa such as alanine (Y84A) or glycine tends to open one end of the MHC binding pocket, allowing a linker (e.g., attached to an peptide epitope) to “thread” through the end of the MHC Class I binding pocket. A Y84A substitution permits greater variation in epitope sizes and allows longer peptides bearing epitope sequences to fit into the binding pocket and be presented by the MAPP. As with aa position 84 substitutions that open one end of the MHC-H binding pocket, substitution of an alanine or glycine at position 167 or its equivalent (e.g., a W167A or W167G substitutions or their equivalent) open the other end of the MHC binding pocket, creating a groove that permits greater variation (e.g., longer length) in the peptide epitopes that may be present. In an embodiment, the HLA-A heavy chain polypeptide of a MAPP comprises the Y84A and A236C substitutions. In an embodiment, the HLA-A heavy chain polypeptide of a MAPP comprises the Y84C and A139C substitutions. When aas 84 and 139 are both cysteines, they may form an intrachain disulfide bond which can stabilize the MHC Class I protein, thereby permitting translation and excretion of the MAPP by eukaryotic cells (e.g., CHO cells), even when not loaded with an peptide epitope. When position 84 is a C residue, it can also form an interchain disulfide bond with a linker attached to the N-terminus of a β2M polypeptide (e.g., epitope-GCGGS(G4S) (SEQ ID NO:87 (where the G45 may be repeated 1-10 times)-mature β2M polypeptide (e.g., lacking its signal sequence), see
The Table below lists some MHC heavy chain sequence modifications that may be incorporated into a MAPP.
(g) β2-Microglobin Polypeptides and Their Combination with MHC-H Polypeptides
A β2M polypeptide of a MAPP 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 aa 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% aa sequence identity to a β2M aa sequence depicted in
In some cases, a MHC Class I polypeptide comprises an aa substitution replacing another aa with a cysteine (Cys) residue. Such cysteine residues can form a disulfide bond with a cysteine residue present in a second polypeptide chain.
In some cases, a β2M polypeptide sequence and an MHC-H polypeptide sequence comprise naturally occurring cysteines substituted for another aa. Such cysteines permit formation of a disulfide bond between those sequences. For example, in some cases MAPPs may contain a disulfide bond between one of following pairs of residues in a HLA β2M (see
Separately, or in addition to, the pairs of cysteine residues in a β2M and HLA Class I heavy chain polypeptide that may be used to form interchain disulfide bonds between the first and second polypeptides of a MAPP (discussed above), the HLA-heavy chain of a MAPP or its epitope conjugate may be substituted with cysteines to form an intrachain disulfide bond between a cysteine substituted into the carboxyl end portion of the al helix and a cysteine in the amino end portion of the α2-1 helix. Such disulfide bonds stabilize the MAPP and permit its cellular processing and excretion from eukaryotic cells in the absence of a bound peptide epitope (or null peptide). In one embodiment, a disulfide bond is formed between the carboxyl end portion of the al helix is from about aa position 79 to about aa position 89 and the amino end portion of the α2-1 helix is from about aa position 134 to about aa position 144 of the MHC Class I heavy chain (the aa positions are determined based on the sequence of the heavy chains without their leader sequence (see, e.g.,
In another embodiment, an intrachain disulfide bond may be formed in a MHC-H sequence of a MAPP between a cysteine substituted into the region between aa positions 79 and 89 and a cysteine substituted into the region between aa positions 134 and 144 of the sequences given in
In an embodiment, the β2M polypeptide sequence of a MAPP comprises a mature β2M polypeptide sequence (aas 21-119) of any one of NP_004039.1, NP_001009066.1, NP_001040602.1, NP_776318.1, or NP_033865.2 (SEQ ID NOs: 1 to 5).
In some cases, a HLA Class I heavy chain polypeptide of a MAPP or its epitope conjugate comprises any one of the HLA-A, -B, -C, -E, -F, or -G sequences in
In an embodiment, the β2M polypeptide of a MAPP comprises a mature β2M polypeptide sequence (aas 21-119) of any one of the sequences in
In an embodiment, a MAPP comprises a mature β2M polypeptide sequence (e.g., aas 21-119 of any one of the sequences in
In an embodiment, a MAPP has a β2M peptide sequence comprising aas 21-119 of a β2M sequence in
Each occurrence of aa cluster 1, aa cluster 2, aa cluster 3, aa cluster 4, aa cluster 5, and aa cluster 6 is independently selected to be 1-5 aa residues, wherein the aa residues are each selected independently from i) any naturally occurring (proteogenic) aa or ii) any naturally occurring aa except proline or glycine.
In an embodiment where the MHC Class I heavy chain is an HLA-A chain:
In some cases, the β2M polypeptide sequence of a MAPP comprises the aa sequence:
In some cases, such as where a MAPP comprises a presenting complex with an epitope and β2M sequence on one of the presenting complex 1st sequence and the presenting complex 2nd sequence, with the MHC-H sequences on the other of presenting complex 1st sequence and the presenting complex 2nd sequence, one or more disulfide bond may be used to link the presenting complex 1st sequence and the presenting complex 2nd sequence (see, e.g.,
A MAPP may comprise one or more immunomodulatory polypeptides or “MODs”. MODs that are suitable for inclusion in a MAPP of the present disclosure include, but are not limited to, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, IL-23, CD7, CD30L, CD40, CD70, CD80, (B7-1), CD83, CD86 (B7-2), HVEM (CD270), ILT3 (immunoglobulin-like transcript 3), ILT4 (immunoglobulin-like transcript 4), Fas ligand (FasL), ICAM (intercellular adhesion molecule), ICOS-L (inducible costimulatory ligand), JAG1 (CD339), lymphotoxin beta receptor, 3/TR6, OX40L (CD252), PD-L1, PD-L2, TGF-β1, TGF-β2, TGF-β3, 4-1BBL, and fragments of any thereof, such as ectodomain fragments, capable of engaging and signaling through their cognate receptor). Some MOD polypeptides suitable for inclusion in a MAPP of the present disclosure, and their “co-MODs (“co-immunomodulatory polypeptides” or cognate costimulatory receptors) include polypeptide sequences with T cell modulatory activity from the protein pairs recited in the following table:
In some cases, the MOD is selected from an IL-2 polypeptide, a 4-1BBL polypeptide, a B7-1 polypeptide; a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, a TGFI3 polypeptide, and a PD-L2 polypeptide. In some cases, the MAPP or duplex MAPP comprises two different MODs, such as an IL-2 MOD or IL-2 variant MOD polypeptide and either a CD80 or CD86 MOD polypeptide. In another instance, the MAPP or duplex MAPP comprises an IL-2 MOD or IL-2 variant MOD polypeptide and a PD-L1 MOD polypeptide. In some case MODs, which may be the same or different, are present in a MAPP or duplex MAPP in tandem. When MODs are presented in tandem, their sequences are immediately adjacent to each other on a single polypeptide, either without any intervening sequence or separated by only a linker polypeptide (e.g., no MHC sequences or epitope sequences intervene). The MOD polypeptide may comprise all or part of the extracellular portion of a full-length MOD. Thus, for example, the MOD can in some cases exclude one or more of a signal peptide, a transmembrane domain, and an intracellular domain normally found in a naturally-occurring MOD. Unless stated otherwise, a MOD present in a MAPP or duplex MAPP does not comprise the signal peptide, intracellular domain, or a sufficient portion of the transmembrane domain to anchor a substantial amount (e.g., more than 5% or 10%) of a MAPP or duplex MAPP into a mammalian cell membrane.
In some cases, a MOD suitable for inclusion in a MAPP comprises all or a portion of (e.g., an extracellular portion of) the aa sequence of a naturally-occurring MOD. In other instances, a MOD suitable for inclusion in a MAPP is a variant MOD that comprises at least one aa substitution compared to the aa sequence of a naturally-occurring MOD. In some instances, a variant MOD exhibits a binding affinity for a co-MOD that is lower than the affinity of a corresponding naturally-occurring MOD (e.g., a MOD not comprising the aa substitution(s) present in the variant) for the co-MOD. Suitable variations in MOD polypeptide sequence that alter affinity may be identified by scanning (making aa substitution e.g., alanine substitutions or “alanine scanning” or charged residue changes) along the length of a peptide and testing its affinity. Once key aa positions altering affinity are identified those positions can be subject to a vertical scan in which the effect of one or more aa substitutions other than alanine are tested.
a. MODs and Variant MODs with Reduced Affinity
A MOD can comprise a wild-type amino acid sequence, or can comprise one or more amino acid substitutions, insertions, and/or deletions relative to a wild-type amino acid sequence. The immunomodulatory polypeptide can comprise only the extracellular portion of a full-length immunomodulatory polypeptide. Alternatively, a MOD can comprise all or a portion of (e.g., an extracellular portion of) the amino acid sequence of a naturally occurring MOD polypeptide.
Variant MODs comprise at least one amino acid substitution, addition and/or deletion as compared to the amino acid sequence of a naturally occurring immunomodulatory polypeptide. As noted above, in some instances a variant MOD exhibits a binding affinity for a co-MOD that is lower than the affinity of a corresponding naturally occurring MOD (e.g., an immunomodulatory polypeptide not comprising the amino acid substitution(s) present in the variant) for the co-MOD.
MOD polypeptides and variants, including reduced affinity variants, of proteins such as PD-L1, CD80, CD86, 4-1BBL and IL-2 are described in the published literature, e.g., published PCT application WO2020132138A1, the disclosure of which as it pertains to immunomodulatory polypeptides and specific variant immunomodulatory polypeptides of PD-L1, CD80, CD86, 4-1BBL, IL-2 are expressly incorporated herein by reference, including specifically paragraphs [00260]-[00455] of WO2020132138A1.
Suitable immunomodulatory domains that exhibit reduced affinity for a co-immunomodulatory domain can have from 1 aa to 20 aa differences from a wild-type immunomodulatory domain. For example, in some cases, a variant MOD present in a MAPP may include a single aa substitution compared to a corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may include 2 aa substitutions compared to a corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may include 3 or 4 aa substitutions compared to a corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may include 5 or 6 aa substitutions compared to a corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may include 7, 8, 9 or 10 aa substitutions compared to a corresponding reference (e.g., wild-type) MOD. A variant MOD present in a MAPP may include 11-15 or 15-20 aa substitutions compared to a corresponding reference (e.g., wild-type) MOD.
As discussed above, a variant MOD suitable for inclusion in a MAPP may exhibit reduced affinity for a cognate co-MOD, compared to the affinity of a corresponding wild-type MOD for the cognate co-MOD. In some cases, a variant MOD present in a MAPP has a binding affinity for a cognate co-MOD that is from 100 nM to 100 μM. For example, in some cases, a variant MOD present in a MAPP has a binding affinity for a cognate co-MOD that is from about 100 nM to about 200 nM, from about 200 nM to about 300 nM, from about 300 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 20 μM, from about 20 μM to about 30 μM, from about 30 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.
Binding affinity between a MOD polypeptide sequence and its cognate co-MOD polypeptide can be determined by bio-layer interferometry (BLI) using the purified MOD polypeptide sequence and purified cognate co-MOD polypeptide, following the procedure set forth in published PCT Application WO 2020/132138 A1.
b. IL-2 and Its Variants
As one non-limiting example, a MOD or variant MOD present in a MAPP is an IL-2 or variant IL-2 polypeptide. In some cases, a variant MOD present in a MAPP is a variant IL-2 polypeptide. Wild-type IL-2 binds to an IL-2 receptor (IL-2R). A wild-type IL-2 aa sequence can be as follows:
Wild-type IL2 binds to an IL2 receptor (IL2R) on the surface of a cell. An IL2 receptor is in some cases a heterotrimeric polypeptide comprising an alpha chain (IL-2Rα; also referred to as CD25), a beta chain (IL-2Rβ; also referred to as CD122) and a gamma chain (IL-2Rγ; also referred to as CD132). Amino acid sequences of human IL-2Rα, IL2Rβ, and IL-2Rγ are provided in the accompanying sequence listing as SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98, respectively, and are also provided in, for example, U.S. Patent Pub. No. 20200407416.
In some cases, a variant IL-2 polypeptide exhibits reduced binding affinity to one or more of the IL-2Rα, IL2Rβ, and/or IL-2Rγ chains of human IL-2R, compared to the binding affinity of an IL-2 polypeptide comprising the aa sequence set forth in SEQ ID NO:95. For example, in some cases, a variant IL-2 polypeptide binds to one or more of the IL-2Rα, IL2Rβ, and/or IL-2Rγ chains of human IL-2R with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less, than the binding affinity of an IL-2 polypeptide comprising the aa sequence set forth in SEQ ID NO:95 for the α,β, and/or γ chains of IL-2R (e.g., an IL-2R comprising polypeptides comprising the aa sequence set forth in SEQ ID NOs:96-98), when assayed under the same conditions.
For example, IL-2 variants with a substitution of phenylalanine at position 42 (e.g., with an alanine), exhibit substantially reduced binding to the IL-2Rα chain, in which case the variant may reduce the activation of Tregs. IL-2 variants with a substitution of histidine at position 16 (e.g., with an alanine) exhibit reduced binding to the IL2Rβ chain, thereby reducing the likelihood of a MAPP binding to non-target T cells by virtue of off-target binding of the IL-2 MOD. Some IL-2 variants, e.g., those with substitutions of the F42 and H16 amino acids, exhibit substantially reduced binding to the IL-2Rα chain and also reduced binding to the IL2Rβ chain. See, e.g., Quayle, et al., Clin Cancer Res; 26(8) Apr. 15, 2020.
In some cases, a variant IL-2 polypeptide has a single aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO:95. In some cases, a variant IL-2 polypeptide has from 2 to 10 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO:95. In some cases, a variant IL-2 polypeptide has 2, 3, 4, 5, 6, 7, 8, 9 or 10 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO:95. In some cases, a variant IL-2 polypeptide has 2 or 3 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO:95.
Suitable variant IL-2 polypeptide sequences include polypeptide sequences comprising an aa sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) aa sequence identity to at least 80 (e.g., 90, 100, 110, 120, 130 or 133) contiguous aas of SEQ ID NO:95. Potential amino acids where substitutions may be introduced include one or more of the following positions:
(i) position 15, where the aa is other than E (e.g., A);
(ii) position 16, where the aa is other than H (e.g., A, T, N, C, Q, M, V or W);
(iii) position 20 is an aa other than D (e.g., A);
(iv) position 42, where the aa is other than F (e.g., A, M, P, S, T, Y, V or H);
(v) position 45, where the aa is other than Y (e.g., A);
(vi) position 88, where the aa is other than N (e.g., A or R);
(vii) position 126, where the aa is other than Q (e.g., A);
Combinations of the above substitutions include (H16X, F42X), (D20X, F42X), (E15X, D20X, F42X), (an H16X, D20X, F42X), (H16X, F42X, R88X), (H16X, F42X, Q126X), (D20X, F42X, Q126X), (D20X, F42X, and Y4X), (H16X, D20X, F42X, and Y45X), (D20X, F42X, Y45X, Q126X), (H16X, D20X, F42X, Y45X, Q126X), where X is the substituted aa, optionally chosen from the following: positions 15, 20, 45, 126—A; position 16—A or T, or also N, C, Q, M, V or W; position 42—A, or also M, P, S, T, Y, V or H; position 88—A or R.
IL-2 variants include polypeptides having at least 90% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 90, 100, 110, 120, or 130) contiguous aas of SEQ ID NO:95, wherein the aa at position 16 is an aa other than H. In one case, the position of H16 is substituted by Asn, Cys, Gln, Met, Val, or Trp. In one case, the position of H16 is substituted by Ala. In another case, the position of H16 is substituted by Thr. Additionally, or alternatively, IL-2 variants include polypeptides having at least 90% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 90, 100, 110, 120, or 130) contiguous aas of SEQ ID NO:95, wherein the aa at position 42 is an aa other than F. In one case, the position of F42 is substituted by Met, Pro, Ser, Thr, Trp, Tyr, Val, or His. In one case, the position of F42 is substituted by Ala.
IL-2 variants include polypeptides comprising an aa sequence comprising all or part of human IL-2 polypeptide having a substitution at position H16 and/or F42 (e.g., H16A and/or F42A substitutions).
IL-2 variants include polypeptides having at least 90% or at least 95% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 100, 110, 120, or 130) contiguous aas of SEQ ID NO:95, wherein the aa at position 16 is an aa other than H and the aa at position 42 is other than F. In one case, the position of H16 is substituted by Ala or Thr and the position of F42 is substituted by Ala or Thr. In one case, the position of H16 is substituted by Ala and the position of F42 is substituted by Ala (an H16A and F42A variant). In a second case, the position of H16 is substituted by Thr and the position of F42 is substituted by Ala (an H16T and F42A variant). In a third case, the position of H16 is substituted by Ala and the position of F42 is substituted by Thr (an H16A and F42T variant). In a fourth case, the position of H16 is substituted by Thr and the position of F42 is substituted Thr Ala (an H16T and F42T variant). As noted above, such variants will exhibit reduced binding to both the human IL-2Rα chain and IL2Rβ chain.
In any of the wild-type or variant IL-2 sequences provided herein, the cysteine at position 125 may be substituted with an aa other than cystine, such as alanine (a C125A substitution). In addition to any stability provided by the substitution, it may be employed where, for example, an epitope containing peptide or additional peptide is to be conjugated to a cysteine residue elsewhere in a MAPP, thereby avoiding competition from the C125 of the IL-2 MOD sequence.
c. Fas Ligand (FasL) and its Variants
In some cases, a wild-type and/or a variant Fas Ligand (FasL) polypeptide sequence is present as a MOD in a MAPP. FasL is a homomeric type-II transmembrane protein in the tumor necrosis factor (TNF) family. FasL signals by trimerization of the Fas receptor in a target cell, which forms a death-inducing complex leading to apoptosis of the target cell. Soluble FasL results from matrix metalloproteinase-7 (MMP-7) cleavage of membrane-bound FasL at a conserved site.
In an embodiment, a wt. Homo sapiens FasL protein has the sequence MQQPFNYPYP QIYWVDSSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPP PPPLPPPPPP PPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG MFQLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L, (SEQ ID NO:99), NCBI Ref. Seq. NP_000630.1, UniProtKB-P48023 where residues 1-80 are cytoplasmic, 81-102 are the transmembrane domain and aas 103-281 are extracellular (ectodomain). In some cases, a FasL polypeptide suitable for inclusion in a MAPP comprises an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to a contiguous stretch of at least 150 aas, at least 170, at least 180 aas, at least 200 aas, at least 225 aas, at least 250 aas, at least 270 aas, at least 280, or all aas of the aa sequence of SEQ ID NO:99).
A Fas receptor can have the sequence MLGIWTLLPL VLTSVARLSS KSVNAQVTDI NSKGLELRKT VTTVETQNLE GLHHDGQFCH KPCPPGERKA RDCTVNGDEP DCVPCQEGKE YTDKAHFSSK CRRCRLCDEG HGLEVEINCT RTQNTKCRCK PNFFCNSTVC EHCDPCTKCE HGIIKECTLT SNTKCKEEGS RSNLGWLCLL LLPIPLIVWV KRKEVQKTCR KHRKENQGSH ESPTLNPETV AINLSDVDLS KYITTIAGVM TLSQVKGFVR KNGVNEAKID EIKNDNVQDT AEQKVQLLRN WHQLHGKKEA YDTLIKDLKK ANLCTLAEKI QTIILKDITS DSENSNFRNE IQSLV, (SEQ ID NO:100) NCBI Reference Sequence: NP_000034.1, UniProtKB-P25445, where aas 26-173 form the ectodomain (extracellular domain), aas 174-190 form the transmembrane domain, and 191-335 the cytoplasmic domain The ectodomain may be used to determine binding affinity with FasL.
A FasL polypeptide suitable for inclusion in a MAPP may comprise an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence: IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMMSYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK (SEQ ID NO:101); and has a length of about 150 aas, including 148, 149, 150, 151, or 152 aas.
In some cases, a FasL polypeptide suitable for inclusion in a MAPP comprises an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to a contiguous stretch of at least 50 aas, at least 160 aas, at least 170, at least 175, or all of the aas the following aa sequence: QLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L (SEQ ID NO:102). Suitable variant FasL polypeptide sequences include polypeptide sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 140 contiguous aa (e.g., at least 150, at least 160, at least 170, or at least 175 contiguous aa) of SEQ ID NO:102) (e.g., which have at least one aa substitution, deletion or insertion).
In some cases, a variant FasL polypeptide (e.g., comprising a variant of SEQ ID NO:101 or SEQ ID NO:102) exhibits reduced binding affinity to a mature Fas receptor sequence (e.g., a FasL receptor comprising all or part of the polypeptide set forth in SEQ ID NO:100, such as its ectodomain), compared to the binding affinity of an FasL polypeptide comprising the aa sequence set forth in SEQ ID NO:101 or SEQ ID NO:102. For example, in some cases, a variant FasL polypeptide (e.g., comprising a variant of SEQ ID NO:102) binds an Fas receptor (e.g., comprising all or part of the polypeptides set forth in SEQ ID NO:100, such as its ectodomains), with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less, than the binding affinity of an FasL polypeptide comprising the aa sequence set forth in SEQ ID NO:99 or 102.
d. PD-L1 and its Variants
As one non-limiting example, a MOD or variant MOD present in a MAPP is a PD-L1 or variant PD-L1 polypeptide. Wild-type PD-L1 binds to PD1.
A wild-type human PD-L1 polypeptide can comprise the following aa sequence: MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKICLT LSPST (SEQ ID NO:103); where aas 1-18 form the signal sequence, aas 19-127 form the Ig-like V-type or IgV domain, and 133-225 for the Ig-like C2 type domain
A wild-type human PD-L1 ectodomain aa sequence can comprise the following aa sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO:104); where aas 1-109 form the Ig-like V-type or “IgV” domain, and aas 115-207 for the Ig-like C2 type domain.
A wild-type human PD-L1 ectodomain aa sequence can also comprise the following aa sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNER LNVSIKI(SEQID NO:105); where aas 1-109 form the Ig-like V-type or “IgV” domain, and aas 115-207 for the Ig-like C2 type domain. See e.g., NCBI Accession and version 3BIK_A, which includes an N-terminal alanine as its first aa.
A wild-type PD-L1 IgV domain, suitable for use as a MOD may comprise aa 18 and aas IgV aas 19-127 of SEQ ID NO:103, and a carboxyl terminal stabilization sequences, such as for instance the last seven aas (bolded and italicized) of the sequence: A FTVTVPKDLY VVEYGSNMT I ECKFPVEKQL DLAALIVYWE MEDKNIIQFV HGEEDLKTQH SSYRQRARLL KDQLSLGNAA LQITDVKLQD AGVYRCMISY GGADYKRITV KVNAPYAAAL HEH (SEQ ID NO:106). Where the carboxyl stabilizing sequence comprises a histidine (e.g., a histidine approximately 5 residues to the C-terminal side of the Tyr (Y) appearing as aa 117 of SEQ ID NO:106) to about aa 122, the histidine may form a stabilizing electrostatic bond with the backbone amide at aas 82 and 83 (bolded and italicized in SEQ ID NO:103 (Q107 and L106 of SEQ ID NO:103). As an alternative, a stabilizing disulfide bond may be formed by substituting one of aas 82 or 83) (Q107 and L106 of SEQ ID NO:103) and one of aa residues 121, 122, or 123 (equivalent to aa positions 139-141 of SEQ ID NO:103).
A wild-type PD-1 polypeptide can comprise the following aa sequence: PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO:107).
In some cases, a variant PD-L1 polypeptide (e.g., a variant of SEQ ID NO:104 or PD-L1's IgV domain) exhibits reduced binding affinity to PD-1 (e.g., a PD-1 polypeptide comprising the aa sequence set forth in SEQ ID NO:107), compared to the binding affinity of a PD-L1 polypeptide comprising the aa sequence set forth in SEQ ID NO:103 or SEQ ID NO:104. For example, in some cases, a variant PD-L1 polypeptide binds PD-1 (e.g., a PD-1 polypeptide comprising the aa sequence set forth in SEQ ID NO:107) with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less than the binding affinity of a PD-L1 polypeptide comprising the aa sequence set forth in SEQ ID NO:103 or SEQ ID NO:104.
e. CD80 and its Variants
In some cases, a wild-type and/or a variant CD80 MOD polypeptide sequence is present as a MOD in a MAPP of the present disclosure. Wild-type CD80 and variant CD80 MOD polypeptides bind to CD28 which acts as their receptor.
A wild-type aa sequence of the ectodomain of human CD80 can be as follows: 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:108). See NCBI Reference Sequence: NP_005182.1. The aa sequence of the IgV domain of a wild-type human CD80 can be as follows:
which is aas 1-104 of SEQ ID NO:108.
A wild-type CD28 aa sequence can be as follows: MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (SEQ ID NO:110).
A wild-type CD28 aa sequence can be as follows: MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSW KHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRS (SEQ ID NO:111)
A wild-type CD28 aa sequence can be as follows: MLRLLLALNL FPSIQVTGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S (SEQ ID NO:112).
Variant CD80 polypeptides suitable as a MOD in a MAPP of the present disclosure may exhibit reduced binding affinity to CD28, compared to the binding affinity of a CD80 polypeptide comprising the aa sequence set forth in SEQ ID NO:108, or the IgV domain sequence SEQ ID NO:109, for CD28. A variant CD80 MOD polypeptide may bind CD28 with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less, than the binding affinity of a CD80 polypeptide comprising the aa sequence set forth in SEQ ID NO:108 for CD28 (e.g., a CD28 polypeptide comprising the aa sequence set forth in one of SEQ ID NO:110, SEQ ID NO:111, or SEQ ID NO:112).
CD80 ectodomain variants suitable for use as a MOD in a MAPP include those polypeptides with at least one aa substitution having at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to SEQ ID NO:108, or the IgV domain sequence SEQ ID NO:109.
CD80 ectodomain variants suitable for use as a MOD in a MAPP include those polypeptides having at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to SEQ ID NO:108, or the IgV domain sequence SEQ ID NO:109, and which have at least one (e.g., at least two, or at least three) aa substitutions.
CD80 ectodomain variants suitable for use as a MOD in a MAPP include those polypeptides having at least 90% (e.g., at least 95%, 98%, or 99%) aa sequence identity to at least 80 (e.g., at least 90, 100, 104, 120, 150, 180, 200, or 208) contiguous aas of SEQ ID NO:108, or least 80 (e.g., at least 90, 100, or 104) contiguous aas of the IgV domain sequence of SEQ ID NO:109.
f. CD86 and its Variants
In some cases, a wild-type and/or a variant CD86 MOD polypeptide sequence is present as a MOD in a MAPP of the present disclosure. Wild-type CD86 and variant CD86 MOD polypeptides bind to CD28 which acts as their receptor as discussed for CD80 MOD polypeptides.
A wild-type aa sequence of the ectodomain of human CD86 can be as follows:
The aa sequence of the IgV domain of a wild-type human CD86 can be as follows:
Variant CD86 polypeptides suitable as a MOD in a MAPP may exhibit reduced binding affinity to CD28, compared to the binding affinity of a CD86 polypeptide comprising the aa sequence set forth in SEQ ID NO:113 or SEQ ID NO:114 for CD28. A variant CD86 MOD polypeptide may bind CD28 with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less, than the binding affinity of a CD86 polypeptide comprising the aa sequence set forth in SEQ ID NO:113 or SEQ ID NO:114 for CD28 (e.g., a CD28 polypeptide comprising the aa sequence set forth in one of SEQ ID NO:110, SEQ ID NO:111, or SEQ ID NO:112).
CD86 ectodomain variants suitable for use as a MOD in a MAPP include those polypeptides with at least one aa substitution having at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to SEQ ID NO:113, or the IgV domain sequence SEQ ID NO:114.
CD86 ectodomain variants suitable for use as a MOD in a MAPP include those polypeptides having at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to SEQ ID NO:113, or the IgV domain sequence SEQ ID NO:114, and which have at least one (e.g., at least two, or at least three) aa substitutions.
CD86 ectodomain variants suitable for use as a MOD in a MAPP include those polypeptides with at least one aa substitution having at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 80 (e.g., at least 90, 100, or 109, 120, 150, 180, 200, or 224) contiguous aas of SEQ ID NO:113, or at least 80 (e.g., at least 90, 100, 104) contiguous aas of the IgV domain sequence SEQ ID NO:114.
g. 4-1BBL and its Variants
In some cases, a wild-type and/or a variant 4-1BBL MOD polypeptide sequence is present as a MOD in a MAPP of the present disclosure. Wild-type 4-1BBL binds to 4-1BB (CD137).
A wild-type 4-1BBL aa sequence can be as follows: MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:115). NCBI Reference Sequence: NP_003802.1, where aas 29-49 are a transmembrane region.
In some cases, a variant 4-1BBL polypeptide is a variant of the tumor necrosis factor (TNF) homology domain (THD) of human 4-1BBL. A wild-type aa sequence of the THD of human 4-1BBL can comprise, e.g., one of SEQ ID NOs:116-118, as follows:
A wild-type 4-1BB aa sequence can be as follows: MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL (SEQ ID NO:119).
A variant 4-1BBL polypeptide exhibits reduced binding affinity to 4-1BB, compared to the binding affinity of a 4-1BBL polypeptide comprising the aa sequence set forth in one of SEQ ID NOs:116-118. For example, a variant 4-1BBL polypeptide may bind 4-1BB with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less, than the binding affinity of a 4-1BBL polypeptide comprising the aa sequence set forth in one of SEQ ID NOs:116-118 for a 4-1BB polypeptide (e.g., a 4-1BB polypeptide comprising the aa sequence set forth in SEQ ID NO:119), when assayed under the same conditions.
h. Anti-CD28
In some cases, antibodies or antibody sequences directed against CD28 (e.g., an anti-CD28 antibody, an anti-body fragment binding CD28, or an scFv, nanobody, or diabody binding to CD28) may be employed as a MOD in a MAPP. 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.
Anti-CD28 antibodies or anti-CD28 sequences may be included in MAPPs in the absence of any other MOD sequences. Alternatively, antibodies or antibody sequences directed against CD28 by be incorporated into a MAPP along with one or more additional MODs, or variant MODs. In an embodiment, A MAPP comprises one or more (e.g., two) anti-CD28 antibody or anti-CD28 sequences along with one or more (e.g., two) 4-1BBL MODs or variant MODs, such as those described above. In an embodiment, A MAPP comprises one or more (e.g., two) anti-CD28 antibody or anti-CD28 sequences along with one or more (e.g., two) IL-2 MODs or variant IL-2 MODs, such as those described above. For example, the substitutions in the variant IL-2 MOD may include H16A or H16T along with an F42A or F42T substitution. By way of example, a MAPP may comprise one or more (e.g., two) anti-CD28 antibody or anti-CD28 sequences (e.g., an anti-CD28 scFv) along with one or more variant IL-2 MODs comprising H16A and/or F42A substitutions.
In some cases, an anti-CD28 antibody suitable for inclusion in a MAPP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence:
In some cases, an anti-CD28 antibody suitable for inclusion in a MAPP 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: QWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPY TFGGGTKLEIKR (SEQ ID NO:475); 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 in a MAPP 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:
In some cases, an anti-CD28 antibody suitable for inclusion in a MAPP 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:
In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:454) and has a length of 15 amino acids.
As noted above, a MAPP can include a linker sequence (aa, peptide, or polypeptide linker sequence) or “linker” interposed between any two elements of a MAPP, e.g., an epitope and an MHC polypeptide; between an MHC polypeptide and an Ig Fc polypeptide; between a first MHC polypeptide and a second MHC polypeptide; etc. Although termed “linkers,” sequences employed for linkers may also be placed at the N- and/or C-terminus of a MAPP polypeptide to, for example, stabilize the MAPP polypeptide or protect it from proteolytic degradation.
Suitable polypeptide linkers (also referred to as “spacers”) are known in the art and can be readily selected and can be of any of a number of suitable lengths, e.g., from 2 to 50 aa in length, e.g., from 2 aa to 10 aa, from l0aa to 20 aa, 20 aa to 30 aa, from 30 aa to 40aa, from 40aa to 50aa, or longer than 50aa. In embodiments, 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, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 aa in length. Linkers can be generally classified into three groups, i.e., flexible, rigid and cleavable. See, e.g., Chen et al. (2013) Adv. Drug Deliv. Rev. 65:1357; and Klein et al. (2014) Protein Engineering, Design & Selection 27:325. Unless stated otherwise, the linkers employed in the MAPPs of this disclosure are not the cleavable linkers generally known in the art.
Polypeptide linkers in the MAPP may include, for example, polypeptides that comprise, consist essentially of, or consists of: i) Gly and Ser; ii) Ala and Ser; iii) Gly, Ala, and Ser; iv) Gly, Ser, and Cys (e.g., a single Cys residue); v) Ala, Ser, and Cys (e.g., a single Cys residue); and vi) Gly, Ala, Ser, and Cys (e.g., a single Cys residue). Exemplary linkers may comprise glycine polymers, glycine-serine polymers, glycine-alanine polymers; alanine-serine polymers (including, for example polymers comprising the sequences GSGGS (SEQ ID NO:120) or GGGS (SEQ ID NO:121), any of which may be repeated from 1 to 10 times (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times); and other flexible linkers known in the art. Glycine and glycine-serine polymers can both be used as both Gly and Ser are relatively unstructured and therefore can serve as a neutral tether between components. Glycine polymers access significantly more phi-psi space than even alanine, and are much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers may also comprise an aa sequence comprising, but not limited to, GGSG (SEQ ID NO:122), GGSGG (SEQ ID NO:123), GSGSG (SEQ ID NO:124), GSGGG (SEQ ID NO:125), GGGSG (SEQ ID NO:126), GSSSG (SEQ ID NO:127), any of which may be repeated from 1 to 10 times (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), or combinations thereof, and the like. Linkers can also comprise the sequence Gly(Ser)4 (SEQ ID NO:128) or (Gly)4Ser (SEQ ID NO:129), either of which may be repeated from 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In one embodiment the linker comprises the aa sequence AAAGG (SEQ ID NO:130), which may be repeated from 1 to 10 times.
Rigid polypeptide linkers comprise a sequence of 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. Rigid polypeptide linkers thus may be employed where it is desired to minimize the interaction between the domains of the MAPP. Rigid peptide linkers include peptide linkers rich in proline, and peptide linkers having an inflexible helical structure, such as an α-helical structure. Examples of rigid peptide linkers include, e.g., (EAAAK)n (SEQ ID NO:547), A(EAAAK)nA (SEQ ID NO:548), A(EAAAK)nALEA(EAAAK)nA (SEQ ID NO:549), (Lys-Pro)n, (Glu-Pro)n, (Thr-Pro-Arg)n, and (Ala-Pro)n 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 linkers comprising EAAAK (SEQ ID NO:550) include EAAAK (SEQ ID NO:550), (EAAAK)2 (SEQ ID NO:551), (EAAAK)3 (SEQ ID NO:552), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO:553), and AEAAAKEAAAKA (SEQ ID NO:554). Non-limiting examples of suitable rigid linkers comprising (AP)n include PAPAP (SEQ ID NO:555; also referred to herein as “(AP)2”); APAPAPAP (SEQ ID NO:556; also referred to herein as “(AP)4”); APAPAPAPAPAP (SEQ ID NO:557; also referred to herein as “(AP)6”); APAPAPAPAPAPAPAP (SEQ ID NO:558; also referred to herein as “(AP)8”); and APAPAPAPAPAPAPAPAPAP (SEQ ID NO:559; also referred to herein as “(AP)10”). Non-limiting examples of suitable rigid linkers comprising (KP)n include KPKP (SEQ ID NO:560; also referred to herein as “(KP)2”); KPKPKPKP (SEQ ID NO:561; also referred to herein as “(KP)4”); KPKPKPKPKPKP (SEQ ID NO:562; also referred to herein as “(KP)6”); KPKPKPKPKPKPKPKP (SEQ ID NO:563; also referred to herein as “(KP)8”); and KPKPKPKPKPKPKPKPKPKP (SEQ ID NO:564; also referred to herein as “(KP)10”). Non-limiting examples of suitable rigid linkers comprising (EP)n include EPEP (SEQ ID NO:565; also referred to herein as “(EP)2”); EPEPEPEP (SEQ ID NO:566; also referred to herein as “(EP)4”); EPEPEPEPEPEP (SEQ ID NO:567; also referred to herein as “(EP)6”); EPEPEPEPEPEPEPEP (SEQ ID NO:568; also referred to herein as “(EP)8”); and EPEPEPEPEPEPEPEPEPEP (SEQ ID NO:569; also referred to herein as “(EP)10”).
As with other linker sequences, rigid peptide linkers may be interposed between any two elements of a MAPP. Rigid peptide linkers find particular use in joining MOD polypeptide sequences to other elements of a MAPP. In particular, rigid peptide linkers may be employed to link a MOD polypeptide sequence to the carboxy terminus of frame work polypeptides (position 3 and/or 3′) or a dimerization polypeptide (positions 5 and/or 5′) of a duplex MAPP. For example, a MOD polypeptide comprising an immunoglobulin CH2CH3 multimerization sequence may comprise a rigid peptide linker and a MOD (e.g., a wild type variant IL-2 or PD-L1 MOD) at position 3 and/or 3′ (See e.g.,
In some cases, a linker polypeptide, present in a polypeptide of a MAPP includes a cysteine residue that can form a disulfide bond with a cysteine residue present in another polypeptide of the MAPP. In some cases, for example, the linker comprises an aa sequence selected from CGGGS (SEQ ID NO:570), GCGGS (SEQ ID NO:571), GGCGS (SEQ ID NO:572), GGGCS (SEQ ID NO:573), and GGGGC (SEQ ID NO:574) with the rest of the linker comprised of Gly and Ser residues (e.g., GGGGS (SEQ ID NO:339) units that may be repeated from 1 to 10 times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). Cysteine containing linkers may also be selected from the sequences GCGASGGGGSGGGGS (SEQ ID NO:131), the sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO:132) or the sequence GCGGSGGGGSGGGGS (SEQ ID NO:133).
Accordingly, the linker to which an epitope is attached may be from about 5 to about 50 aas in length. The linker to which an epitope may be attached may, for example be from about 5 to about 50 aas in length and comprise more than 50% Gly and Ser residues with one cysteine residue. The linker to which an epitope may be attached may be from about 5 to about 50 aas in length and comprise more than 50% (Gly)4S repeats with one optional cysteine residue. The linker to which an epitope may be attached may be a (Gly)4S sequence repeated from 3 to 8 (e.g., 3 to 7) times, optionally having one aa replaced by a cysteine residue.
A variety of peptide epitopes (also referred to herein as “epitopes”) may be present in a MAPP or higher order complexes of MAPPs (such as duplex MAPPs of the present disclosure), and presentable to a TCR on the surface of a T cell.
An epitope-presenting peptide can have a length of from about 4 aas to about 25 aas (aa), e.g., the epitope can have a length of from 4 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to 20 aa, or from 20 aa to 25 aa. For example, an epitope present in a MAPP of the present disclosure can have a length of 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 some cases, an epitope-presenting peptide present in a MAPP of the present disclosure has a length of from 5 aa to 10 aa, e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.
Epitope presenting peptides (or simply epitopes) may be derived from a variety of self and non-self antigens, depending upon the nature of the MAPP and its desired use. Non-self antigens (including neoantigens) may be incorporated into MAPPs for the treatment or prophylaxis of, for example, cancers, allergies, and viral and bacterial diseases. Self-antigens may be incorporated into MAPPs for the treatment or prophylaxis of, for example, cancers, infections and allergies.
A peptide epitope present in a MAPP (e.g., a duplex MAPP) is designed to be specifically bound by a target T cell that has a T cell receptor (“TCR”) that is specific for the epitope and which specifically binds the peptide epitope of the MAPP. An epitope-specific T cell thus binds a peptide epitope having a reference aa sequence, but substantially does not bind an epitope that differs from the reference aa sequence. For example, an epitope-specific T cell binds a peptide epitope having a reference aa sequence, and binds an epitope that differs from the reference aa 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 a peptide 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 M.
a. Epitope-Presenting Peptides in MAPPs with Class I MHC Presenting Sequences and Presenting Complexes
Among the epitopes that may be bound and presented to a TCR by a MAPP with Class I MHC presenting sequences or presenting complexes are cancer antigens, and antigens from infectious agents (e.g., viral or bacterial agents). Where T Cell dysregulation (e.g., CD8+ T cell dysregulation) result in over reaction to allergens, the epitope that may be presented include epitopes of allergens and autoantigens, e.g., those associated with Type 1 diabetes (T1D) or celiac disease. For example, an allergen may be selected from protein or non-proteins components of: nuts (e.g., tree and/or peanuts), glutens, pollens, eggs (e.g., chicken, Gallus domesticus eggs), shellfish, soy, fish, and insect venoms (e.g., bee and/or wasp venom antigens). Similarly, where dysregulation of CD8+ T reg cells and self-reactive CD8+ effector T cells result in autoimmune diseases the epitope presented may be from a protein associated with, for example, multiple sclerosis, Rasmussen's encephalitis, paraneoplastic syndromes. Grave's disease (GD), systemic lupus erythematosus (SLE), aplastic anemia (AA), or vitiligo.
(i) Cancer Epitopes
Suitable epitopes include in a MAPP or higher order MAPP complexes such as duplexes MAPPs, but are not limited to, epitopes present in a cancer-associated antigen. Cancer-associated antigens are known in the art; see, e.g., Cheever et al. (2009) Clin. Cancer Res. 15:5323. Cancer-associated antigens include, but are not limited to, α-folate receptor; carbonic anhydrase IX (CAIX); CD19; CD20; CD22; CD30; CD33; CD44v7/8; carcinoembryonic antigen (CEA); epithelial glycoprotein-2 (EGP-2); epithelial glycoprotein-40 (EGP-40); folate binding protein (FBP); fetal acetylcholine receptor; ganglioside antigen GD2; Her2/neu; IL-13R-a2; kappa light chain; LeY; L1 cell adhesion molecule; melanoma-associated antigen (MAGE); MAGE-A1; mesothelin; MUC1; NKG2D ligands; oncofetal antigen (h5T4); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); tumor-associate glycoprotein-72 (TAG-72); vascular endothelial growth factor receptor-2 (VEGF-R2) (see, e.g., Vigneron et al. (2013) Cancer Immunity 13:15; and Vigneron (2015) BioMed Res. Int'l Article ID 948501); and epidermal growth factor receptor (EGFR) vIII polypeptide (see, e.g., Wong et al. (1992) PNAS. USA 89:2965; and Miao et al. (2014) PLoSOne 9:e94281).
In some cases, a suitable peptide epitope is a peptide fragment of from about 4 aas (aa) to about 20 aa (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, or 20 aa) in length of 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 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 alpha-fetoprotein (AFP) 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 Al) polypeptide, a cytochrome P450 1B1 (CYP1B1) 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 (PDGFβ) polypeptide, a MAD-CT-2 polypeptide, or a Fos-related antigen-1 (FOSL) polypeptide. In some cases, a human papilloma virus (HPV) antigen is specifically excluded. In some cases, an alpha-feto protein (AFP) antigen is specifically excluded. In some cases, a Wilms tumor-1 (WT1) antigen is specifically excluded.
Amino acid sequences of cancer-associated antigens are known in the art; see, e.g., MUC1 (GenBank CAA56734); LMP2 (GenBank CAA47024); EGFRvIII (GenBank NP_001333870); HER-2/neu (GenBank AAI67147); MAGE-A3 (GenBank AAH11744); p53 (GenBank BAC16799); NY-ESO-1 (GenBank CAA05908); PSMA (GenBank AAH25672); CEA (GenBank AAA51967); melan/MART1 (GenBank NP_005502); Ras (GenBank NP_001123914); gp100 (GenBank AAC60634); bcr-abl (GenBank AAB60388); tyrosinase (GenBank AAB60319); survivin (GenBank AAC51660); PSA (GenBank CAD54617); hTERT (GenBank BAC11010); SSX (GenBank NP_001265620); Eph2A (GenBank NP_004422); PAP (GenBank AAH16344); ML-IAP (GenBank AAH14475); EpCAM (GenBank NP_002345); ERG (TMPRSS2 ETS fusion) (GenBank ACA81385); PAX3 (GenBank AAI01301); ALK (GenBank NP_004295); androgen receptor (GenBank NP_000035); cyclin B1 (GenBank CA099273); MYCN (GenBank NP_001280157); RhoC (GenBank AAH52808); TRP-2 (GenBank AAC60627); mesothelin (GenBank AAH09272); PSCA (GenBank AAH65183); MAGE Al (GenBank NP_004979); CYP1B1 (GenBank AAM50512); PLAC1 (GenBank AAG22596); BORIS (GenBank NP_001255969); ETV6 (GenBank NP_001978); NY-BR1 (GenBank NP_443723); SART3 (GenBank NP_055521); carbonic anhydrase IX (GenBank EAW58359); PAXS (GenBank NP_057953); OY-TES1 (GenBank NP_115878); sperm protein 17 (GenBank AAK20878); LCK (GenBank NP_001036236); HMW-MAA (GenBank NP_001888); AKAP-4 (GenBank NP_003877); SSX2 (GenBank CAA60111); XAGE1 (GenBank NP_001091073; XP_001125834; XP_001125856; and XP_001125872); B7H3 (GenBank NP_001019907; XP_947368; XP_950958; XP_950960; XP_950962; XP_950963; XP_950965; and XP_950967); LGMN1 (GenBank NP_001008530); TIE-2 (GenBank NP_000450); PAGE4 (GenBank NP_001305806); VEGFR2 (GenBank NP_002244); MAD-CT-1 (GenBank NP_005893 NP_056215); FAP (GenBank NP_004451); PDGFβ (GenBank NP_002600); MAD-CT-2 (GenBank NP_001138574); and FOSL (GenBank NP_005429). These polypeptides are also discussed in, e.g., Cheever et al. (2009) Clin. Cancer Res. 15:5323, and references cited therein; Wagner et al. (2003) J. Cell. Sci. 116:1653; Matsui et al. (1990) Oncogene 5:249; Zhang et al. (1996) Nature 383:168.
(a) Alpha Feto Protein (AFP)
MAPPs or higher order MAPP complexes such as duplex MAPPs described herein may comprise a peptide presenting an epitope of alpha-feto protein (AFP), which has been associated with hepatocellular carcinoma, pancreatic cancer, stomach cancer, colorectal cancer, hepatoblastoma, and an ovarian yolk sac tumor. The AFP epitope may be presented in the context of a Class I MHC presenting sequence. The AFP epitope may be presented in the context of a Class I MHC presenting complex. The Class I MHC may be a) an aa sequence having at least 95% aa sequence identity to the HLA-A*0101, HLA-A*0201, HLA-A*1101, HLA-A*2301, HLA-A*2402, HLA-A*2407, HLA-A*3303, or HLA-A*3401; b) an aa sequence having at least 95% aa sequence identity to the HLA-B*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601, or HLA-B*5301; or c) an aa sequence having at least 95% aa sequence identity to the HLA-C*0102, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLA-C*0702,HLA-C*0801, or HLA-C*1502 depicted in
AFP peptides for inclusion in a MAPP include, but are not limited to, AITRKMAAT (SEQ ID NO:134); AYTKKAPQL (SEQ ID NO:135); LLNQHACAV (SEQ ID NO:136); KLVLDVAHV (SEQ ID NO:137); FMNKFIYEI (SEQ ID NO:138); SIPLFQVPE (SEQ ID NO:139); LLNFTESRT (SEQ ID NO:140); FVQEATYKF (SEQ ID NO:141); ATYKEVSKM (SEQ ID NO:142); KEVSKMVKD (SEQ ID NO:143); RHNCFLAHK (SEQ ID NO:144); ATAATCCQL (SEQ ID NO:145); YIQESQALA (SEQ ID NO:146); QLTSSELMAI (SEQ ID NO:147); KLSQKFTKV (SEQ ID NO:148); KELRESSLL (SEQ ID NO:149); SLVVDETYV (SEQ ID NO:150); ILLWAARYD (SEQ ID NO:151); KIIPSCCKA (SEQ ID NO:152); CRGDVLDCL (SEQ ID NO:153); QQDTLSNKI (SEQ ID NO:154); TMKQEFLINL (SEQ ID NO:155); NLVKQKPQI (SEQ ID NO:156); AVIADFSGL (SEQ ID NO:157); LLACGEGAA (SEQ ID NO:158); LACGEGAAD (SEQ ID NO:159); KAPQLTSSEL (SEQ ID NO:160); YICSQQDTL (SEQ ID NO:161); TECCKLTTL (SEQ ID NO:162); CTAEISLADL (SEQ ID NO:163); VTKELRESSL (SEQ ID NO:164); IMSYICSQQD (SEQ ID NO:165); TRTFQAITV (SEQ ID NO:166); FQKLGEYYL (SEQ ID NO:167); RVAKGYQEL (SEQ ID NO:168); SYQCTAEISL (SEQ ID NO:169);KQEFLINLV (SEQ ID NO:170); MKWVESIFL (SEQ ID NO:171); PVNPGVGQC (SEQ ID NO:172); AADIIIGHL (SEQ ID NO:173); QVPEPVTSC (SEQ ID NO:174); TTLERGQCII (SEQ ID NO:175); KMAATAATC (SEQ ID NO:176); QAQGVALQTM (SEQ ID NO:177); FQAITVTKL (SEQ ID NO:178); LLEKCFQTE (SEQ ID NO:179); VAYTKKAPQ (SEQ ID NO:180); KYIQESQAL (SEQ ID NO:181); GVALQTMKQ (SEQ ID NO:182); GQEQEVCFA (SEQ ID NO:183); SEEGRHNCFL (SEQ ID NO:184); RHPFLYAPTI (SEQ ID NO:185); TEIQKLVLDV (SEQ ID NO:186); RRHPQLAVSV (SEQ ID NO:187); GEYYLQNAFL (SEQ ID NO:188); NRRPCFSSLV (SEQ ID NO:189); LQTMKQEFLI (SEQ ID NO:190); IADFSGLLEK (SEQ ID NO:191); GLLEKCCQGQ (SEQ ID NO:192); TLSNKITEC (SEQ ID NO:193); LQDGEKIMSY (SEQ ID NO:194); GLFQKLGBY (SEQ ID NO:195); NEYGIASILD (SEQ ID NO:196); KMVKDALTAI (SEQ ID NO:197); FLASFVHEY (SEQ ID NO:198); AQFVQEATY (SEQ ID NO:199); EYSRRHPQL (SEQ ID NO:411); AYEEDRETF (SEQ ID NO:412; SYANRRPCF (SEQ ID NO:413); CFAEEGQKL (SEQ ID NO:414); RSCGLFQKL (SEQ ID NO:415); IFLIFLLNF (SEQ ID NO:416); KPEGLSPNL (SEQ ID NO:417); and GLSPNLNRFL (SEQ ID NO:418).
In some cases, the AFP peptide present in a TMMP of the present disclosure presents an HLA-A*2402-restricted epitope. Non-limiting examples of AFP peptides that present an HLA-A*2402-restricted epitope are: KYIQESQAL (SEQ ID NO:181); EYYLQNAFL (SEQ ID NO:419); AYTKKAPQL (SEQ ID NO:135); EYSRRHPQL (SEQ ID NO:411); RSCGLFQKL (SEQ ID NO:415) and AYEEDRETF (SEQ ID NO:412).
In some cases, the AFP peptide present in a TMMP of the present disclosure is KYIQESQAL (SEQ ID NO:181). In some cases, the AFP peptide present in a TMMP of the present disclosure is EYYLQNAFL (SEQ ID NO:419. In some cases, the AFP peptide present in a TMMP of the present disclosure is AYTKKAPQL (SEQ ID NO:135). In some cases, the AFP peptide present in a TMMP of the present disclosure is EYSRRHPQL (SEQ ID NO:411). In some cases, the AFP peptide present in a TMMP of the present disclosure is RSCGLFQKL (SEQ ID NO:415).
In some cases, the AFP peptide present in a TMMP of the present disclosure presents an HLA-A*0201-restricted epitope. Non-limiting examples of AFP peptides that present an HLA-A*0201-restricted epitope are: FMNKFIYEI (SEQ ID NO:138); and GLSPNLNRFL (SEQ ID NO:418).
(b) Wilms Tumor Antigen (WT-1)
MAPPs or higher order MAPP complexes such as duplex MAPPs described herein may comprise a peptide presenting an epitope of Wilms Tumor-1 protein, which has been associated with myeloid leukemia, myeloma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, colorectal cancer, breast cancer, Wilms tumor, mesothelioma, soft tissue sarcoma, neuroblastoma, and nephroblastoma. The WT-1 epitope may be presented in the context of a Class I MHC presenting sequence. The WT-1 epitope may be presented in the context of a Class I MHC presenting complex. The Class I MHC may be a) an aa sequence having at least 95% aa sequence identity to the HLA-A*0101, HLA-A*0201, HLA-A*1101, HLA-A*2301, HLA-A*2402, HLA-A*2407, HLA-A*3303, or HLA-A*3401; b) an aa sequence having at least 95% aa sequence identity to the HLA-B*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601, or HLA-B*5301; or c) an aa sequence having at least 95% aa sequence identity to the HLA-C*0102, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLA-C*0702,HLA-C*0801, or HLA-C*1502 depicted in
WT-1 peptides for inclusion in a MAPP include, but are not limited to, NLMNLGATL (SEQ ID NO:200), NYMNLGATL (SEQ ID NO:201), CMTWNQMNLGATLKG (SEQ ID NO:202), WNQMNLGATLKGVAA (SEQ ID NO:203), CMTWNYMNLGATLKG (SEQ ID NO:204), WNYMNLGATLKGVAA (SEQ ID NO:205), TWNQMNLGATLKGV (SEQ ID NO:206), TWNQMNLGATLKGVA (SEQ ID NO:207), CMTWNLMNLGATLKG (SEQ ID NO:208), MTWNLMNLGATLKGV (SEQ ID NO:209), TWNLMNLGATLKGVA (SEQ ID NO:210), WNLMNLGATLKGVAA (SEQ ID NO:211), MNLGATLK (SEQ ID NO:212), MTWNYMNLGATLKGV SEQ ID NO:213), TWNYMNLGATLKGVA (SEQ ID NO:214), CMTWNQMNLGATLKGVA (SEQ ID NO:215), CMTWNLMNLGATLKGVA (SEQ ID NO:216), CMTWNYMNLGATLKGVA (SEQ ID NO:217), GYLRNPTAC (SEQ ID NO:218), GALRNPTAL (SEQ ID NO:219), YALRNPTAC (SEQ ID NO:220), GLLRNPTAC (SEQ ID NO:221), RYRPHPGAL (SEQ ID NO:222), YQRPHPGAL (SEQ ID NO:223), RLRPHPGAL (SEQ ID NO:224), RIRPHPGAL (SEQ ID NO:225), QFPNHSFKHEDPMGQ (SEQ ID NO:226), HSFKHEDPY (SEQ ID NO:227), QFPNHSFKHEDPM (SEQ ID NO:228), QFPNHSFKHEDPY (SEQ ID NO:229), KRPFMCAYPGCNK (SEQ ID NO:230), KRPFMCAYPGCYK (SEQ ID NO:231), FMCAYPGCY (SEQ ID NO:232), FMCAYPGCK (SEQ ID NO:233), KRPFMCAYPGCNKRY (SEQ ID NO:234), SEKRPFMCAYPGCNK (SEQ ID NO:235), KRPFMCAYPGCYKRY (SEQ ID NO:236), NLMNLGATL (SEQ ID NO:237), VLDFAPPGA (SEQ ID NO:439); RMFPNAPYL (SEQ ID NO:440); YMFPNAPYL (SEQ ID NO:441); SLGEQQYSV (SEQ ID NO:442); CYTWNQMNL (SEQ ID NO:443); CMTWNQMNL (SEQ ID NO:444); NQMNLGATL (SEQ ID NO:445); and NYMNLGATL (SEQ ID NO:238).
In some cases, the WT-1 peptide present in a TMMP of the present disclosure presents an HLA-A*2402-restricted epitope. WT-1 peptides that present an HLA-A*2402-restricted epitope include, e.g., CMTWNQMN (SEQ ID NO:446); NYMNLGATL (SEQ ID NO:238) (WT-1 239-247; Q240Y); CYTWNQMNL (SEQ ID NO:443) (WT-1 235-243); CMTWNQMNL (SEQ ID NO:444) (WT-1 235-243); NQMNLGATL (SEQ ID NO:445) (WT-1 239-247); and NLMNLGATL (SEQ ID NO:237) (WT-1239-247; Q240L).
In some cases, the WT-1 peptide present in a TMMP of the present disclosure presents an HLA-A*0201-restricted epitope. WT-1 peptides that present an HLA-A*0201-restricted epitope include, e.g., VLDFAPPGA (SEQ ID NO:439) (WT-1 37-45); RMFPNAPYL (SEQ ID NO:440) (WT-1 126-134); YMFPNAPYL (SEQ ID NO:441) (WT-1 126-134; R126Y); SLGEQQYSV (SEQ ID NO:442) (WT-1 187-195); and NLMNLGATL (SEQ ID NO:237) (WT-1 239-247; Q240L).
(c) Human Papilloma Virus I (HPV)
MAPPs or higher order MAPP complexes such as duplex MAPPs described herein may comprise a peptide presenting an epitope of human papilloma virus (HPV), which has been associated with cervical cancer, prostate cancer, or ovarian cancer. HPV epitopes may be presented in the context of a Class I MHC presenting sequence. The HPV epitope may be presented in the context of a Class I MHC presenting complex. The Class I MHC may be a) an aa sequence having at least 95% aa sequence identity to the HLA-A*0101, HLA-A*0201, HLA-A*1101, HLA-A*2301, HLA-A*2402, HLA-A*2407, HLA-A*3303, or HLA-A*3401; b) an aa sequence having at least 95% aa sequence identity to the HLA-B*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601, or HLA-B*5301; or c) an aa sequence having at least 95% aa sequence identity to the HLA-C*0102, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLA-C*0702,HLA-C*0801, or HLA-C*1502 depicted in
HPV peptide epitopes include, but are not limited to, those from the E6 and E7 gene products:
In some cases, the epitope is HPV16E7/82-90 (LLMGTLGIV; SEQ ID NO:251). In some cases, the epitope is HPV16E7/86-93 (TLGIVCPI; SEQ ID NO:252). In some cases, the epitope is HPV16E7/11-20 (YMLDLQPETT; SEQ ID NO:254). In some cases, the epitope is HPV16E7/11-19 (YMLDLQPET; SEQ ID NO:255). See, e.g., Ressing et al. ((1995) J. Immunol. 154:5934) for additional suitable HPV epitopes.
In some cases, a suitable HPV peptide is an HPV E6 peptide that binds HLA-A24 (e.g., is an HLA-A2401-restricted epitope). Non-limiting examples include: VYDFAFRDL (SEQ ID NO:241); CYSLYGTTL (SEQ ID NO:266); EYRHYCYSL (SEQ ID NO:420); KLPQLCTEL (SEQ ID NO:239); DPQERPRKL (SEQ ID NO:399); HYCYSLYGT (SEQ ID NO:421); DFAFRDLCI (SEQ ID NO:422); LYGTTLEQQY (SEQ ID NO:423); HYCYSLYGTT (SEQ ID NO:424); EVYDFAFRDL (SEQ IDNO:425); EYRHYCYSLY (SEQ ID NO:426); VYDFAFRDLC (SEQ ID NO:427); YCYSIYGTTL (SEQ ID NO:428); VYCKTVLEL (SEQ ID NO:429); VYGDTLEKL (SEQ ID NO:430); and LTNTGLYNLL (SEQ ID NO:431).
In some cases, a suitable HPV peptide is selected from the group consisting of: DLQPETTDL (SEQ ID NO:432); TLHEYMLDL (SEQ ID NO:245); TPTLHEYML (SEQ ID NO:433); RAHYNIVTF (SEQ ID NO:248); GTLGIVCPI (SEQ ID NO:434); EPDRAHYNI (SEQ ID NO:435); QLFLNTLSF (SEQ ID NO:436); FQQLFLNTL (SEQ ID NO:437); and AFQQLFLNTL (SEQ IDNO:438).
In some cases, a suitable HPV peptide presents an HLA-A*2401-restricted epitope. Nonlimiting examples of HPV peptides presenting an HLA-A*2401-restricted epitope are: VYDFAFRDL (SEQ ID NO:241); RAHYNIVTF (SEQ ID NO:248); CDSTLRLCV (SEQ ID NO:249); and LCVQSTHVDI (SEQ ID NO:250). In some cases, an HPV peptide suitable for inclusion in a TMMP of the present disclosure is VYDFAFRDL (SEQ ID NO:241). In some cases, an HPV peptide suitable for inclusion in a TMMP of the present disclosure is RAHYNIVTF (SEQ ID NO:248). In some cases, an HPV peptide suitable for inclusion in a TMMP of the present disclosure is CDSTLRLCV (SEQ IDNO:249). In some cases, an HPV peptide suitable for inclusion in a TMMP of the present disclosure is LCVQSTHVDI (SEQ ID NO:250).
(d) Hepatitis B Virus (HBV)
MAPPs or higher order MAPP complexes such as duplex MAPPs described herein may comprise a peptide presenting an epitope of hepatitis B virus (HBV), which has been associated with hepatocellular carcinoma. HBV epitopes may be presented in the context of a Class I MHC presenting complex. The Class I MHC may be a) an aa sequence having at least 95% a sequence identity to the HLA-A*0101, HLA-A*0201, HLA-A*1101, HLA-A*2301, HLA-A*2402, HLA-A*2407, HLA-A*3303, or HLA-A*3401; b) an aa sequence having at least 95% aa sequence identity to the HLA-B*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601, or HLA-B*5301; or c) an aa sequence having at least 95% aa sequence identity to the HLA-C*0102, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLA-C*0702,HLA-C*0801, or HLA-C*1502 depicted in
HBV peptide epitopes include, but are not limited to, FLPSDFFPSV (SEQ ID NO:256), GLSRYVARLG (SEQ ID NO:257), KLHLYSHPI (SEQ ID NO:258), FLLSLGIHL (SEQ ID NO:259), ALMPLYACI (SEQ ID NO:260), SLYADSPSV (SEQ ID NO:261), STLPETTVV (SEQ ID NO:262), LIMPARFYPK (SEQ ID NO:263), AIMPARFYPK (SEQ ID NO:264), YVNVNMGLK (SEQ ID NO:265), MQWNSTALHQALQDP (SEQ ID NO:267), LLDPRVRGL (SEQ ID NO:268), SILSKTGDPV (SEQ ID NO:269), VLQAGFFLL (SEQ ID NO:270), FLLTRILTI (SEQ ID NO:271), FLGGTPVCL (SEQ ID NO:272), LLCLIFLLV (SEQ ID NO:273), LVLLDYQGML (SEQ ID NO:274), LLDYQGMLPV (SEQ ID NO:275), IPIPSSWAF (SEQ ID NO:276), WLSLLVPFV (SEQ ID NO:277), GLSPTVWLSV (SEQ ID NO:278), SIVSPFIPLL (SEQ ID NO:279), ILSPFLPLL (SEQ ID NO:280), ATVELLSFLPSDFFPSV (SEQ ID NO:281), LPSDFFPSV (SEQ ID NO:282), CLTFGRETV (SEQ ID NO:283), VLEYLVSFGV (SEQ ID NO:284), EYLVSFGVW (SEQ ID NO:285), ILSTLPETTV (SEQ ID NO:286), STLPETTVVRR (SEQ ID NO:287), NVSIPWTHK (SEQ ID NO:288), KVGNFTGLY (SEQ ID NO:289), GLYSSTVPV (SEQ ID NO:290), TLWKAGILYK (SEQ ID NO:291), TPARVTGGVF (SEQ ID NO:292), LVVDFSQFSR (SEQ ID NO:293), GLSRYVARL (SEQ ID NO:294), SIACSVVRR (SEQ ID NO:295), YMDDVVLGA(SEQ ID NO:296), PLGFFPDH (SEQ ID NO:297), QAFTFSPTYK (SEQ ID NO:298), KYTSFPWLL (SEQ ID NO:299), ILRGTSFVYV (SEQ ID NO:300), HLSLRGLFV (SEQ ID NO:301), VLHKRTLGL (SEQ ID NO:302), GLSAMSTTDL (SEQ ID NO:303), CLFKDWEEL (SEQ ID NO:304), and VLGGCRHKL (SEQ ID NO:305).
(ii) Infectious Agents
Suitable epitopes from infectious agents that may be included in MAPPs or higher order MAPP complexes such as duplex MAPPs include, but are not limited to, epitopes present in an infectious virus, bacterium, fungus, protozoan, or helminth disease causing agents, 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, Cytomegalovirus, Epstein Barr virus, Herpes simplex types 1 and 2, Herpes zoster, Human herpesvirus types 6 and 7, hepatitis C virus (HCV, see above), hepatitis B virus (HBV, see above), 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, Orbivirus (Colorado tick fever virus), Rabies virus, Vesicular stomatitis virus, Rubivirus (rubella), Semliki Forest virus, St. Louis encephalitis virus, Venezuelan equine encephalitis 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), 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, 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), 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, Lumpy skin disease virus, Malignant catarrhal fever virus, Menangle virus, 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 MAPP of the present disclosure can include a peptide from any known viral antigen. In some cases, an HPV antigen is specifically excluded. In some cases, an HBV antigen is specifically excluded.
In some cases, the peptide epitope present in a MAPP of the present disclosure presents an epitope specific to an HLA-A, -B, -C, -E, -F or -G allele. In an embodiment, the peptide epitope present in a MAPP presents an epitope restricted to HLA-A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and/or A*3401. In an embodiment, the peptide epitope present in a MAPP presents an epitope restricted to HLA-B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and/or B*5301. In an embodiment, the peptide epitope present in a MAPP presents an epitope restricted to C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*702, C*0801, and/or C*1502.
b. HLA/Peptide Binding Assays
Whether a given peptide (e.g., WT-1 peptide) 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.
(i) Cell-Based Binding Assay
As one example, a cell-based peptide-induced stabilization assay can be used to determine peptide-HLA Class I binding. In this assay, a peptide of interest is allowed to bind to a TAP-deficient cell, i.e., a cell that has defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface Class I molecules. Such cells include, e.g., the human T2 cell line (T2 (174×CEM.T2; American Type Culture Collection (ATCC) No. CRL-1992). Henderson et al. (1992) Science 255:1264. Without efficient TAP-mediated transport of cytosolic peptides into the endoplasmic reticulum, assembled MHC Class I complexes are structurally unstable, and retained only transiently at the cell surface. However, when T2 cells are incubated with an exogenous peptide capable of binding Class I, surface peptide-HLA Class I complexes are stabilized and can be detected by flow cytometry with, e.g., a pan anti-Class I monoclonal antibody. The stabilization and resultant increased life-span of peptide-HLA complexes on the cell surface by the addition of a peptide validates their identity. Analysis can be carried out using flow cytometry, e.g., where the pan-HLA Class I antibody comprises a fluorescent label. Binding of the peptide to various allelic forms of HLA H chains can be tested by genetically modifying the T2 cells to express an allelic HLA H chain of interest.
The following is a non-limiting example of use of a T2 assay to assess peptide binding to HLA A*0201. T2 cells are washed in cell culture medium, and concentrated to 106 cells/ml. Peptides of interest are prepared in cell culture medium and serially diluted providing concentrations of 200 μM, 100 μM, 20 μM and 2 μM. The cells are mixed 1:1 with each peptide dilution to give a final volume of 200 μL and final peptide concentrations of 100 μM, 50 μM, 10 μM and 1 μM. A HLA A*0201 binding peptide, GILGFVFTL, and a non-HLA A*0201-restricted peptide, HPVGEADYF (HLA-B*3501), are included as positive and negative controls, respectively. The cell/peptide mixtures are kept at 37° C. 5% CO2 for ten minutes; then incubated at room temperature overnight. Cells are then incubated for 2 hours at 37° C. and stained with a fluorescently-labeled anti-human HLA antibody. The cells are washed twice with phosphate-buffered saline and analyzed using flow cytometry. The average mean fluorescence intensity (MFI) of the anti-HLA antibody staining is used to measure the strength of binding.
(ii) Biochemical Binding Assay
HLA polypeptides (HLA heavy chain polypeptide complexed with β2M polypeptide) can be tested for binding to a peptide of interest in a cell-free in vitro assay system. For example, a labeled reference peptide (e.g., fluorescently labeled) is allowed to bind to HLA polypeptides (HLA heavy chain polypeptide complexed with β2M polypeptide), to form an HLA-reference peptide complex. The ability of a test peptide of interest to displace the labeled reference peptide from the HLA-reference peptide complex is tested. The relative binding affinity is calculated as the amount of test peptide needed to displace the bound reference peptide. See, e.g., van der Burg et al. (1995) Human Immunol. 44:189.
As another example, a peptide of interest can be incubated with an HLA molecule (HLA heavy chain complexed with a β2M polypeptide), and the stabilization of the HLA/peptide complex can be measured in an immunoassay format. The ability of a peptide of interest to stabilize an HLA molecule is compared to that of a control peptide presenting a known T cell epitope. Detection of stabilization is based on the presence or absence of the native conformation of the HLA/peptide complex, detected using an anti-HLA antibody. See, e.g., Westrop et al. (2009) J. Immunol. Methods 341:76; Steinitz et al. (2012) Blood 119:4073; and U.S. Pat. No. 9,205,144.
(iii) T Cell Activation Assays
Whether a given peptide 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 by assessing T cell response to the peptide-HLA complex. T cell responses that can be measured include, e.g., interferon-gamma (IFNγ) production, cytotoxic activity, and the like.
(iv) ELISPOT Assay
Suitable assays include, e.g., an enzyme linked immunospot (ELISPOT) assay. In this assay, production of IFNγ by CD8+ T cells is measured following with an antigen-presenting cell (APC) that presents a peptide of interest complexed with HLA Class I. Antibody to IFNγ is immobilized on wells of a multi-well plate. APCs are added to the wells, and incubated for a period of time with a peptide of interest, such that the peptide binds HLA Class I on the surface of the APCs. CD8+ T cells specific for the peptide are added to the wells, and the plate is incubated for about 24 hours. The wells are then washed, and any IFNγ bound to the immobilized anti-IFNγ antibody is detected using a detectably labeled anti-IFNγ antibody. A colorimetric assay can be used. For example, the detectably labeled anti-IFNγ antibody can be a biotin-labeled anti-IFNγ antibody, which can be detected using, e.g., streptavidin conjugated to alkaline phosphatase. A BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) solution is added, to develop the assay. The presence of IFNγ-secreting T cells is identified by colored spots. Negative controls include APCs not contacted with the peptide. APCs expressing various HLA H chain alleles can be used to determine whether a peptide of interest effectively binds to a HLA Class I molecule comprising a particular HLA H chain.
(v) Cytotoxicity Assays
Whether a given peptide binds to a particular HLA Class I H chain and, when bound to a HLA Class I complex comprising the H chain, can effectively present an epitope to a TCR, can also be determined using a cytotoxicity assay. A cytotoxicity assay involves incubation of a target cell with a cytotoxic CD8+ T cell. The target cell displays on its surface a peptide/HLA Class I complex comprising a peptide of interest and an HLA Class I molecule comprising an HLA H chain to be tested. The target cells can be radioactively labeled, e.g., with 51Cr. Whether the target cell effectively presents an epitope to a TCR on the cytotoxic CD8+ T cell, thereby inducing cytotoxic activity by the CD8+ T cell toward the target cell, is determined by measuring release of 51Cr from the lysed target cell. Specific cytotoxicity can be calculated as the amount of cytotoxic activity in the presence of the peptide minus the amount of cytotoxic activity in the absence of the peptide.
(vi) Detection of Antigen-Specific T Cells with Peptide-HLA Tetramers
As another example, multimers (e.g., tetramers) of peptide-HLA complexes are generated with fluorescent or heavy metal tags. The multimers can then be used to identify and quantify specific T cells via flow cytometry (FACS) or mass cytometry (CyTOF). Detection of epitope-specific T cells provides direct evidence that the peptide-bound HLA molecule is capable of binding to a specific TCR on a subset of antigen-specific T cells. See, e.g., Klenerman et al. (2002) Nature Reviews Immunol. 2:263.
A polypeptide chain of a MAPP of the present disclosure (e.g., a dimerization or framework polypeptide) may include one or more polypeptides in addition to those described above. Suitable additional polypeptides include epitope tags and affinity domains. The one or more additional polypeptides can be included at the N-terminus of a polypeptide chain of a MAPP of the present disclosure, at the C-terminus of a polypeptide chain of a MAPP of the present disclosure, or within (internal to) a polypeptide chain of a MAPP of the present disclosure.
a. Affinity Tags, Epitope Tags and Affinity Domains
Suitable affinity/epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO:306); FLAG (e.g., DYKDDDDK (SEQ ID NO:307); c-myc (e.g., EQKLISEEDL; SEQ ID NO:308), and the like.
Affinity domains include peptide sequences that can interact with a binding partner, e.g., such as one immobilized on a solid support, useful for identification or purification. DNA sequences encoding multiple consecutive single aas, such as histidine, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high affinity binding to a resin column, such as nickel sepharose. Exemplary affinity domains include HisX5 (HHHHH) (SEQ ID NO:309), HisX6 (HHHHHH) (SEQ ID NO:310), C-myc (EQKLISEEDL) (SEQ ID NO:311), Flag (DYKDDDDK) (SEQ ID NO:312), StrepTag (WSHPQFEK) (SEQ ID NO:313), hemagglutinin, e.g., HA Tag (YPYDVPDYA) (SEQ ID NO:314), glutathione-S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:315), Phe-His-His-Thr (SEQ ID NO:316), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag, WEAAAREACCRECCARA (SEQ ID NO:317), metal binding domains, e.g., zinc binding domains or calcium binding domains such as those from calcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large-subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, calretinin, inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltose binding protein.
b. Targeting Sequences
MAPPs may include, as part of any one or more framework and/or any one or more dimerization polypeptide, a targeting polypeptide or “targeting sequence.” Targeting sequences serve to bind or “localize” MAPPs to cells and/or tissues displaying the protein (or other molecule) to which the targeting sequence binds. Targeting sequences may be located, for example at or near the carboxyl terminal end of a framework or dimerization peptide (e.g., in place of a C-terminal MOD in
In some instances, a targeting sequence present in a MAPP targets an antigen of an infecting organism and/or of an infected cell. Targeting polypeptides may be directed to proteins/epitopes of infectious agents including, but not limited to, viruses, bacteria, fungi, protozoans, and helminths, including those proteins/epitopes of infectious agents that are expressed on cell surfaces. By way of example, cells infected with HPV may express E6 or E7 proteins (described herein as cancer associated epitope) or portions thereof to which the targeting sequence may be directed. A targeting sequence may also be a Cancer Targeting Polypeptide, or “CTP” that is specific for a cancer associated antigen (“CAA”), such as an antigen associated with a non-solid cancer (e.g., a leukemia) and/or solid tumor-associated antigen. In one instance, the targeting sequence is specific for a cancer-associated 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). MAPPs can also be targeted to specific tissues or cell types by using targeting sequences directed toward molecules expressed selectively by cells of the desired tissue.
(a) Cancer Associated Antigens (CAAs)
CAAs that can be targeted with a CTP present in a MAPP or a higher order MAPP complex, such as a duplex MAPP, of the present disclosure 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, 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 ROR1 (type I receptor tyrosine kinase like orphan receptor).
CAAs that can be targeted with a CTP present in a MAPP of the present disclosure also 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, CD28, 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, 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 αVβ3, integrin α5β1, 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 CAA is an antigen that may be targeted with a CTP of a MAPP 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 CAA 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, a CAA that is to be targeted by a CTP is 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, ROR1, TAG-72, tenascin, TRAILR1, TRAILR2, VEGF, VEGFR, and others listed above.
(b) Peptide/HLA Complexes
In some cases, a CTP of a MAPP or a higher order MAPP complex, such as a duplex MAPP, targets a peptide/HLA (pHLA) complex on the surface of a cancer cell, where the peptide is a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen). Cancer-associated peptide antigens are known in the art. In some cases, a cancer-associated peptide is bound to a HLA complex comprising an HLA-A*0201 heavy chain and a β2M polypeptide.
In some cases, the CAA peptide 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 peptide 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 peptide 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, a CAA peptide is a peptide of from about 4 aas (aa) to about 20 aa (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, or 20 aa) in length of any one of the following cancer-associated antigens: a CD28 polypeptide, 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 Al) polypeptide, a cytochrome P450 1B1 (CYP1B1) 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 (RGSS) polypeptide, a squamous cell carcinoma antigen recognized by T cells (SART3) polypeptide, a carbonic anhydrase IX polypeptide, a paired box-5 (PAXS) 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 Akinase 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 (PDGFβ) 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 (WT1) antigen.
For example, in some cases, a CTP present in a MAPP of the present disclosure 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 CAA peptide is a peptide of from about 4 aas (aa) to about 20 aa (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, or 20 aa) in length of a mesothelin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following mesothelin aa sequence: LAGE TGQEAAPLDG VLANPPNISS LSPRQLLGFP CAEVSGLSTE RVRELAVALA QKNVKLSTEQ LRCLAHRLSE PPEDLDALPL DLLLFLNPDA FSGPQACTRF FSRITKANVD LLPRGAPERQ RLLPAALACW GVRGSLLSEA DVRALGGLAC DLPGRFVAES AEVLLPRLVS CPGPLDQDQQ EAARAALQGG GPPYGPPSTW SVSTMDALRG LLPVLGQPII RSIPQGIVAA WRQRSSRDPS WRQPERTILR PRFRREVEKT ACPSGKKARE IDESLIFYKK WELEACVDAA LLATQMDRVN AIPFTYEQLD VLKHKLDELY PQGYPESVIQ HLGYLFLKMS PEDIRKWNVT SLETLKALLE VNKGHEMSPQ VATLIDRFVK GRGQLDKDTL DTLTAFYPGY LCSLSPEELS SVPPSSIWAV RPQDLDTCDP RQLDVLYPKA RLAFQNMNGS EYFVKIQSFL GGAPTEDLKA LSQQNVSMDL ATFMKLRTDA VLPLTVAEVQ KLLGPHVEGL KAEERHRPVR DWILRQRQDD LDTLGLGLQG GIPNGYLVLD LSMQEALSGT PCLLGPGPVL TVLALLLAST LA (SEQ ID NO:318). For example, a mesothelin peptide present in a pHLA complex can be: i) KLLGPHVEGL (SEQ ID NO:319); ii) AFYPGYLCSL (SEQ ID NO:320), which can bind HLA-A*2402/β2M; iii) VLPLTVAEV (SEQ ID NO:321); iv) ELAVALAQK (SEQ ID NO:322); v) ALQGGGPPY (SEQ ID NO:323); vi) FYPGYLCSL (SEQ ID NO:324); vii) LYPKARLAF (SEQ ID NO:325); viii) LLFLLFSLGWVGPSR (SEQ ID NO:326); ix) VNKGHEMSPQAPRRP (SEQ ID NO:327); x) FMKLRTDAVLPLTVA (SEQ ID NO:328); or xi) DAALLATQMD (SEQ ID NO:329).
In some cases, a CAA peptide is a peptide of from about 4 aas (aa) to about 20 aa (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, or 20 aa) in length of a mesothelin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following Her2 (receptor tyrosine-protein kinase erbB2) aa sequence:
In some cases, a CAA peptide is a peptide of from about 4 aas (aa) to about 20 aa (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, or 20 aa) in length of a B-cell maturation protein (BCMP) polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following BCMA aa sequence:
In some cases, a CAA peptide is a peptide of from about 4 aas (aa) to about 20 aa (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, or 20 aa) in length of a mesothelin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following WT-1 aa sequence: MDFLLLQDPA STCVPEPASQ HTLRSGPGCL QQPEQQGVRD PGGIWAKLGA AEASAERLQG RRSRGASGSE PQQMGSDVRD LNALLPAVPS LGGGGGCALP VSGAAQWAPV LDFAPPGASA YGSLGGPAPP PAPPPPPPPP PHSFIKQEPS WGGAEPHEEQ CLSAFTVHFS GQFTGTAGAC RYGPFGPPPP SQASSGQARM FPNAPYLPSC LESQPAIRNQ GYSTVTFDGT PSYGHTPSHH AAQFPNHSFK HEDPMGQQGS LGEQQYSVPP PVYGCHTPTD SCTGSQALLL RTPYSSDNLY QMTSQLECMT WNQMNLGATL KGHSTGYESD NHTTPILCGA QYRIHTHGVF RGIQDVRRVP GVAPTLVRSA SETSEKRPFM CAYPGCNKRY FKLSHLQMHS RKHTGEKPYQ CDFKDCERRF SRSDQLKRHQ RRHTGVKPFQ CKTCQRKFSRSDHLKTHTRT HTGEKPFSCR WPSCQKKFAR SDELVRHHNM HQRNMTKLQL AL (SEQ ID NO:332).
Non-limiting examples of WT-1 peptides include RMFPNAPYL (SEQ ID NO:397), CMTWNQMN (SEQ ID NO:403), CYTWNQMNL (SEQ ID NO:400), CMTWNQMNLGATLKG (SEQ ID NO:202), WNQMNLGATLKGVAA (SEQ ID NO:203), CMTWNYMNLGATLKG (SEQ ID NO:204), WNYMNLGATLKGVAA (SEQ ID NO:205), MTWNQMNLGATLKGV (SEQ ID NO:447), TWNQMNLGATLKGVA (SEQ ID NO:207), CMTWNLMNLGATLKG (SEQ ID NO:208), MTWNLMNLGATLKGV (SEQ ID NO:209), TWNLMNLGATLKGVA (SEQ ID NO:210), WNLMNLGATLKGVAA (SEQ ID NO:211), MNLGATLK (SEQ ID NO:212), MTWNYMNLGATLKGV (SEQ ID NO:213), TWNYMNLGATLKGVA (SEQ ID NO:214), CMTWNQMNLGATLKGVA (SEQ ID NO:215), CMTWNLMNLGATLKGVA (SEQ ID NO:216), CMTWNYMNLGATLKGVA (SEQ ID NO:217), GYLRNPTAC (SEQ ID NO:218), GALRNPTAL (SEQ ID NO:219), YALRNPTAC (SEQ ID NO:220), GLLRNPTAC (SEQ ID NO:221), RYRPHPGAL (SEQ ID NO:222), YQRPHPGAL (SEQ ID NO:223), RLRPHPGAL (SEQ ID NO:224), RIRPHPGAL (SEQ ID NO:225), QFPNHSFKHEDPMGQ (SEQ ID NO:226), HSFKHEDPY (SEQ ID NO:227), QFPNHSFKHEDPM (SEQ ID NO:228), QFPNHSFKHEDPY (SEQ ID NO:229), KRPFMCAYPGCNK (SEQ ID NO:230), KRPFMCAYPGCYK (SEQ ID NO:231), FMCAYPGCY (SEQ ID NO:232), FMCAYPGCK (SEQ ID NO:233), KRPFMCAYPGCNKRY (SEQ ID NO:234), SEKRPFMCAYPGCNK (SEQ ID NO:235), KRPFMCAYPGCYKRY (SEQ ID NO:236), NLMNLGATL (SEQ ID NO:237), NYMNLGATL (SEQ ID NO:238), and those WT-1 peptides recited in section IV.C.7.a.(i)(b).
In some cases, a CAA is a peptide of from about 4 aas (aa) to about 20 aa (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, or 20 aa) in length of an HPV polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to a human papilloma virus (HPV) peptide. An HPV peptide can be a peptide of an HPV E6 polypeptide or an HPV E7 protein. 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:239); E6 26-34 (LQTTIHDII; SEQ ID NO:240); E6 49-57 (VYDFAFRDL; SEQ ID NO:241); E6 52-60 (FAFRDLCIV; SEQ ID NO:242); E6 75-83 (KFYSKISEY; SEQ ID NO:243); E6 80-88 (ISEYRHYCY; SEQ ID NO:244); E7 7-15 (TLHEYMLDL; SEQ ID NO:245); E7 11-19 (YMLDLQPET; SEQ ID NO:246); E7 44-52 (QAEPDRAHY; SEQ ID NO:247); E7 49-57 (RAHYNIVTF (SEQ ID NO:248); E7 61-69 (CDSTLRLCV; SEQ ID NO:249); and E7 67-76 (LCVQSTHVDI; SEQ ID NO:250); E7 82-90 (LLMGTLGIV; SEQ ID NO:251); E7 86-93 (TLGIVCPI; SEQ ID NO:252); E7 92-93 (LLMGTLGIVCPI; SEQ ID NO:253); and those HPV peptides recited herein (see, e.g., section IV.C.7.a.(i).(c)).
In some cases, a CAA 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: MAVTACQGLG FVVSLIGIAG IIAATCMDQW STQDLYNNPV TAVFNYQGLW RSCVRESSGF TECRGYFTLL GLPAMLQAVR ALMIVGIVLG AIGLLVSIFA LKCIRIGSME DSAKANMTLT SGIMFIVSGL CAIAGVSVFA NMLVTNFWMS TANMYTGMGG MVQTVQTRYT FGAALFVGWV AGGLTLIGGV MMCIACRGLA PEETNYKAVS YHASGHSVAY KPGGFKASTG FGSNTKNKKI YDGGARTEDE VQSYPSKHDY V (SEQ ID NO:448). In some cases, a cancer-associated peptide is a peptide of a claudin polypeptide having the amino acid sequence TEDEVQSYPSKHDYV (SEQ ID NO:449) (and having a length of about 15 amino acids) or EVQSYPSKHDYV (SEQ ID NO:450) (and having a length of about 12 amino acids.
In some cases, a CAA 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: QDNCTCPTNK MTVCSPDGPG GRCQCRALGS GMAVDCSTLT SKCLLLKARM SAPKNARTLV RPSEHALVDN DGLYDPDCDP EGRFKARQCN QTSVCWCVNS VGVRRTDKGD LSLRCDELVR THHILIDLRH RPTAGAFNHS DLDAELRRLF RERYRLHPKF VAAVHYEQPT IQIELRQNTS QKAAGDVDIG DAAYYFERDI KGESLFQGRG GLDLRVRGEP LQVERTLIYY LDEIPPKFSM KRLTAGLIAV IVVVVVALVA GMAVLVITNR RKSGKYKKVE IKELGELRKE PSL (SEQ ID NO:451).
(ii) Antibodies
As noted above, in some cases, a CTP present in a MAPP or a higher order MAPP complex, such as a duplex MAPP, of the present disclosure is an antibody or an antigen binding fragment thereof. In some cases, the CTP is an antibody that is specific for a CAA. In some cases, the CTP is an antibody specific for a peptide on the surface of an infected cell (e.g., viral, bacterial, or mycoplasma). In some cases, the CTP 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 fragment of a cancer-associated antigen).
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.
The term “humanized immunoglobulin” as used herein refers to an immunoglobulin comprising portions of immunoglobulins of different origin, wherein at least one portion comprises amino acid sequences of human origin. Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin.
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, F(ab′)2, 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 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).
“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.
“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 in the CDR-table below 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.
Non-limiting examples of CAA-targeted antibodies (or antigen binding fragments thereof) that can be included in a MAPP of the present disclosure include, but are not limited to, abituzumab (anti-CD51), LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20), obinutuzumab (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, a CAA-targeted antibody (or antigen binding fragments thereof) that can be included in a MAPP is a single-chain antibody. In some cases, a CAA-targeted antibody (or antigen binding fragments thereof) that can be included in a MAPP 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 aa 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.
(a) Anti-Her2
In some cases, an anti-Her2 antibody (or antigen binding fragments thereof) useful as a CTP comprises: a) a light chain comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence:
In some cases, an anti-Her2 antibody (or antigen binding fragments thereof) comprises a light chain variable region (VL) present in the light chain aa sequence provided above; and a heavy chain variable region (VH) present in the heavy chain aa sequence provided above. For example, an anti-Her2 antibody can comprise: a) a VL comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence:
and b) a VH comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: EVQLVESGGGLVQPGGSLRLSCA ASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSL RAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:336). In some cases, an anti-Her2 antibody comprises, in order from N-terminus to C-terminus: a) a VH comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAM DYWGQGTLVTVSS (SEQ ID NO:337); b) a linker; and c) a VL comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSA SFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:338). Suitable linker sequences described elsewhere herein and include, e.g., (GGGGS) (SEQ ID NO:339), which may be repeated from 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times).
In some cases, an anti-Her2 antibody (or antigen binding fragments thereof) comprises VL CDR1, VL CDR2, and VL CDR3 present in the light chain aa sequence provided above; and VH CDR1, CDR2, and CDR3 present in the heavy chain aa sequence provided above. In some cases, the VH and VL CDRs are as defined by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); 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. In some cases, the VH and VL CDRs are as defined by Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987). For example, an anti-Her2 antibody (or antigen binding fragments thereof) can comprise a VL CDR1 having the aa sequence RASQDVNTAVA (SEQ ID NO:340); a VL CDR2 having the aa sequence SASFLY (SEQ ID NO:341); a VL CDR3 having the aa sequence QQHYTTPP (SEQ ID NO:342); a VH CDR1 having the aa sequence GFNIKDTY (SEQ ID NO343); a VH CDR2 having the aa sequence IYPTNGYT (SEQ ID NO:344); and a VH CDR3 having the aa sequence SRWGGDGFYAMDY (SEQ ID NO:345).
In some cases, an anti-Her2 antibody (or antigen binding fragments thereof) is a scFv antibody. For example, an anti-Her2 scFv can comprise an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence: EVQLVESGGGLVQPG GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTA YLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGT DFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:346).
As another example, in some cases, an anti-Her2 antibody (or antigen binding fragments thereof) comprises: a) a light chain variable region (VL) comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKL LIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:347); and b) a heavy chain variable region (VH) comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence: EVQLVESGGG LVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSV DRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:348).
In some cases, an anti-Her2 antibody (or antigen binding fragments thereof) useful as a CTP comprises a VL present in the light chain aa sequence provided above; and a VH present in the heavy chain aa sequence provided above. For example, an anti-Her2 antibody can comprise: a) a VL comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKP GKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEI K (SEQ ID NO:349); and b) a VH comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: EVQLVESGGGLVQPGGSLRLS CAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQM NSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO:350).
In some cases, an anti-Her2 antibody (or antigen binding fragments thereof) used as a CTP comprises VL CDR1, VL CDR2, and VL CDR3 present in the light chain aa sequence provided above; and VH CDR1, CDR2, and CDR3 present in the heavy chain aa sequence provided above. In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Chothia 1987). For example, an anti-HER2 antibody can comprise a VL CDR1 having the aa sequence KASQDVSIGVA (SEQ ID NO:351); a VL CDR2 having the aa sequence SASYRY (SEQ ID NO:352); a VL CDR3 having the aa sequence QQYYIYPY (SEQ ID NO:353); a VH CDR1 having the aa sequence GFTFTDYTMD (SEQ ID NO:354); a VH CDR2 having the aa sequence ADVNPNSGGSIYNQRFKG (SEQ ID NO:355); and a VH CDR3 having the aa sequence ARNLGPSFYFDY (SEQ ID NO:356).
In some cases, an anti-Her2 antibody (or antigen binding fragments thereof) is a scFv. For example, in some cases, an anti-Her2 scFv comprises an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence:
(b) Anti-CD19
Anti-CD19 antibodies (and antigen binding fragments thereof) useful as a CTP 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 MAPP of the present disclosure. See e.g., WO 2005/012493.
In some cases, an anti-CD19 antibody (or antigen binding fragments thereof) includes a VL CDR1 comprising the aa sequence KASQSVDYDGDSYLN (SEQ ID NO:358); a VL CDR2 comprising the aa sequence DASNLVS (SEQ ID NO:359); and a VL CDR3 comprising the aa sequence QQSTEDPWT (SEQ ID NO:360). In some cases, an anti-CD19 antibody (or antigen binding fragments thereof) includes a VH CDR1 comprising the aa sequence SYWMN (SEQ ID NO:361); a VH CDR2 comprising the aa sequence QIWPGDGDTNYNGKFKG (SEQ ID NO:362); and a VH CDR3 comprising the aa sequence RETTTVGRYYYAMDY (SEQ ID NO:363). In some cases, an anti-CD19 antibody includes a VL CDR1 comprising the aa sequence KASQSVDYDGDSYLN (SEQ ID NO:364); a VL CDR2 comprising the aa sequence DASNLVS (SEQ ID NO:365); a VL CDR3 comprising the aa sequence QQSTEDPWT (SEQ ID NO:366); a VH CDR1 comprising the aa sequence SYWMN (SEQ ID NO:367); a VH CDR2 comprising the aa sequence QIWPGDGDTNYNGKFKG (SEQ ID NO:368); and a VH CDR3 comprising the aa sequence RETTTVGRYYYAMDY (SEQ ID NO:369).
In some cases, an anti-CD19 antibody (or antigen binding fragments thereof) is a scFv. For example, in some cases, an anti-CD19 scFv comprises an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence: DIQLTQSPAS LAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDF TLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAEL VRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTAD ESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVS (SEQ ID NO:370).
(c) Anti-Mesothelin
Anti-mesothelin antibodies (or antigen binding fragments thereof) useful as a CTP 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 MAPP of the present disclosure. 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, an anti-mesothelin antibody (or antigen binding fragments thereof) comprises: a) a light chain comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNN RPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLGQPKAAPSV TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLS LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS (SEQ ID NO:371); and b) a heavy chain comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence: QVELVQSGAEVKKPGESLKISCKGSGYS FTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTA MYYCARGQLYGGTYMDGWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:372).
In some cases, an anti-mesothelin antibody (or antigen binding fragments thereof) comprises a VL present in the light chain aa sequence provided above; and a VH present in the heavy chain aa sequence provided above. For example, an anti-mesothelin antibody can comprise: a) a VL comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPK LMIYGVNNR PSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTK (SEQ ID NO:373); and b) a VH comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: QVELVQSGAEVKKPGESLKISCKGS GYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKAS DTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO:374).
In some cases, an anti-mesothelin antibody (or antigen binding fragments thereof) comprises VL CDR1, VL CDR2, and VL CDR3 present in the light chain aa sequence provided above; and VH CDR1, CDR2, and CDR3 present in the heavy chain aa sequence provided above. In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, Chothia 1987). For example, an anti-mesothelin antibody (or antigen binding fragments thereof) can comprise a VL CDR1 having the aa sequence TGTSSDIGGYNSVS (SEQ ID NO:375); a VL CDR2 having the aa sequence LMIYGVNNRPS (SEQ ID NO:376); a VL CDR3 having the aa sequence SSYDIESATP (SEQ ID NO:377); a VH CDR1 having the aa sequence GYSFTSYWIG (SEQ ID NO:378); a VH CDR2 having the aa sequence WMGIIDPGDSRTRYSP (SEQ ID NO:379); and a VH CDR3 having the aa sequence GQLYGGTYMDG (SEQ ID NO:380).
An anti-mesothelin antibody can be a scFv. As one non-limiting example, an antimesothelin scFv can comprise the following aa sequence: QVQLQQSGAEVKKPGASV KVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTA YMELSRLRSEDTAVYYCARGRYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSEIV LTQSPATLSLSPGERATISCRASQSVSSNFAWYQQRPGQAPRLLIYDASNRATGIPPRFSGSGSG TDFTLTISSLEPEDFAAYYCHQRSNWLYTFGQGTKVDIK (SEQ ID NO:381), 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 aa sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPN SGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLRRTVVTPRAYYGMDV WGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCQASODISNSL NWYQQKAGKAPKWYDASTLETGVPSRFSGSGSGTDFSFTISSLQPEDIATYYCQQHDNLPLT FGQGTKVEIK (SEQ ID NO:382), 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 MAPP as a targeting sequence comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFT FGPGTKVDIK (SEQ ID NO:452); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QMQLVESGGGVVQPGRSLRLS CTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLE MNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO:453). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., the CDR Table, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., the CDR Table, above; and Chothia 1987).
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence 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: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATG IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO:452); 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: QMQLVESG GGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTI SRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO:453).
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence 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: QMQLVESGGGVVQPGRSLRLSCTASGFT FSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAE DTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO:453); 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: EIVLTQSPGT LSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO:452). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence 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: EIVLTQSPGTLSLSPGERATLSCRASQSVSS SYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPI FTFGPGTKVDIK (SEQ ID NO:452); 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: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGM HWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYC AREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO:453). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYM HWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPL TFGSGTKVEIK (SEQ ID NO:455); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLQQSGPELEKPGASVKISC KASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLS LTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO:456). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., the CDR Table, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., the CDR Table, above; and Chothia 1987).
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence 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: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGV PGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO:455); 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: QVQLQQSGP ELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTV DKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO:456).
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence 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: DIELTQSPAIMSASPGEKVTMTCSASSSVS YMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKH PLTFGSGTKVEIK (SEQ ID NO:455); 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: QVQLQQSGPELEKPGASVKISCKASGYSF TGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSA VYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO:456). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence 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: QVQLQQSGPELEKPGASVKISCKASGYSF TGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSA VYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO:456); 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: DIELTQSPAIMSASPGEK VTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDD ATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO:455). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYN SVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIES ATPVFGGGTKLTVLG (SEQ ID NO:457); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVELVQSGAEVKKPGES LKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQ WSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO:458). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., the CDR Table, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., the CDR Table, above; and Chothia 1987).
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence 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: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRP SGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO:457); 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 MAPP as a targeting sequence 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: DIALTQPASVSGSPGQSITISCTGTSSDIG GYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSY DIESATPVFGGGTKLTVLG (SEQ ID NO:457); 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: QVELVQSGAEVKKPGESLKISCKGSGYS FTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTA MYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO:458). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP as a targeting sequence 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: QVELVQSGAEVKKPGESLKISCKGSGYSF TSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM YYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO:458); 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: DIALTQPASVSGSPGQ SITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQ AEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO:457). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDT SKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO:459); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYT MNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVY YCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO:460). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., the CDR Table, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., the CDR Table, above; and Chothia 1987).
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP 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: DIQMTQSPSSLSASV GDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO:459); 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: QVQLVQSGAEVKKPG ASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMT VDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO:460).
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP 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: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQK SGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFG QGTKLEIK (SEQ ID NO:459); 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: QVQLVQSGAEVKKPGASVKVSCKASGYSFT GYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSL RSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO:460). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-mesothelin antibody suitable for inclusion in a MAPP 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: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWV RQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARG GYDGRGFDYWGQGTLVTVSS (SEQ ID NO:460); 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: DIQMTQSPSSLSASVGDRVTITCSASS SVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWS KHPLTFGQGTKLEIK (SEQ ID NO:459). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
(d) Anti-TROP-2
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 CTP of a MAPP 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 MAPP of the present disclosure as a targeting sequence. 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:461); CDR2 (SASYRYT; SEQ ID NO:462); and CDR3 (QQHYITPLT; SEQ ID NO:463); and ii) heavy chain CDR sequences CDR1 (NYGMN; SEQ ID NO:464); CDR2 (WINTYTGEPTYTDDFKG; SEQ ID NO:465); and CDR3 (GGFGSSYWYFDV; SEQ ID NO:466).
In some cases, an anti-TROP-2 antibody comprises: i) heavy chain CDR sequences CDR1 (TAGMQ; SEQ ID NO:467); CDR2 (WINTHSGVPKYAEDFKG (SEQ ID NO:468); and CDR3 (SGFGSSYWYFDV; SEQ ID NO:469); and ii) light chain CDR sequences CDR1 (KASQDVSTAVA; SEQ ID NO:470); CDR2 (SASYRYT; SEQ ID NO:462); and CDR3 (QQHYITPLT; SEQ ID NO:463).
In some cases, an anti-TROP2 antibody suitable for inclusion in a MAPP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSA SYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO:471); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYG MNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYF CARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO:472). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., the CDR Table, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., the CDR Table, above; and Chothia 1987).
In some cases, an anti-TROP-2 antibody suitable for inclusion in a MAPP 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: DIQLTQSPSSLSASVGD RVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPED FAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO:471); 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: QVQLQQSGSELKKPGASVKVSCKASGYT FTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADD TAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO:472).
In some cases, an anti-TROP-2 antibody suitable for inclusion in a MAPP 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: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAP KLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO:471); 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: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAP GQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSS YWYFDVWGQGSLVTVSS (SEQ ID NO:472). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-TROP-2 antibody suitable for inclusion in a MAPP 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: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQ APGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFG SSYWYFDVWGQGSLVTVSS (SEQ ID NO:472); 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: DIQLTQSPSSLSASVGDRVSITCKAS QDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQ HYITPLTFGAGTKVEIK (SEQ ID NO:471). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-TROP2 antibody suitable for inclusion in MAPP comprises: a) VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLL IYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO:473); and b) VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAG MQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYY CARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO:474). In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., the CDR Table, above; and Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., the CDR Table, above; and Chothia 1987).
In some cases, an anti-TROP-2 antibody suitable for inclusion in a MAPP 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: DIQMTQSPSSLSAS VGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSL QPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO:473); 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: QVQLVQSGAEVKKPGASVKVSCKASGY TFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSE DTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO:474).
In some cases, an anti-TROP-2 antibody suitable for inclusion in a MAPP 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: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGK APKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO:473); 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: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQA PGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGS SYWYFDVWGQGTLVTVSS (SEQ ID NO:474). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
In some cases, an anti-TROP-2 antibody suitable for inclusion in a MAPP 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: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQ APGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFG SSYWYFDVWGQGTLVTVSS (SEQ ID NO:474); 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: DIQMTQSPSSLSASVGDRVTITCKASQ DVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQH YITPLTFGQGTKLEIK (SEQ ID NO:473). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n, 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:454) and has a length of 15 amino acids.
(e) Anti-BCMA
Anti-BCMA (B-cell maturation antigen) antibodies (or antigen binding fragments thereof) 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 MAPP of the present disclosure. See, e.g., WO 2014/089335; and US 2019/0153061.
In some cases, an anti-BCMA antibody (or antigen binding fragments thereof) comprises: a) a light chain comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence: QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGS KSGSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPDSKQSNNKYAASSYLSLTPEQWKSH RSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:383); and b) a heavy chain comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the following aa sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLE WVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWT PFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:384).
In some cases, an anti-BCMA (or antigen binding fragments thereof) comprises a VL present in the light chain aa sequence provided above; and a VH present in the heavy chain aa sequence provided above. For example, an anti-BCMA antibody can comprise: a) a VL comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence:
In some cases, an anti-BCMA antibody (or antigen binding fragments thereof) comprises VL CDR1, VL CDR2, and VL CDR3 present in the light chain aa sequence provided above; and VH CDR1, CDR2, and CDR3 present in the heavy chain aa sequence provided above. In some cases, the VH and VL CDRs are as defined by Kabat (see, e.g., Kabat 1991). In some cases, the VH and VL CDRs are as defined by Chothia (see, e.g., Chothia 1987).
For example, an anti-BCMA antibody (or antigen binding fragments thereof) can comprise a VL CDR1 having the aa sequence SSNIGSNT (SEQ ID NO:387), a VL CDR2 having the aa sequence NYH, a VL CDR3 having the aa sequence AAWDDSLNGWV (SEQ ID NO:388)), a VH CDR1 having the aa sequence GFTFGDYA (SEQ ID NO:389), a VH CDR2 having the aa sequence SRSKAYGGTT (SEQ ID NO:390), and a VH CDR3 having the aa sequence ASSGYSSGWTPFDY (SEQ ID NO:391).
An anti-BCMA antibody can be a scFv. As one non-limiting example, an anti-BCMA scFv can comprise the following aa sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHW VRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRV TITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO:392), or the sequence: QVQLVQSGAEVKKPGSS VKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTST AYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGG GSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO:480).
As another example, an anti-BCMA scFv can comprise the following aa sequence:
In some cases, an anti-BCMA antibody can comprise a VL CDR1 having the amino acid sequence SASQDISNYLN (SEQ ID NO:481); a VL CDR2 having the amino acid sequence YTSNLHS (SEQ ID NO:482); a VL CDR3 having the amino acid sequence QQYRKLPWT (SEQ ID NO:483); a VH CDR1 having the amino acid sequence NYWMH (SEQ ID NO:484); a VH CDR2 having the amino acid sequence ATYRGHSDTYYNQKFKG (SEQ ID NO:485); and a VH CDR3 having the amino acid sequence GAIYNGYDVLDN (SEQ ID NO:486).
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: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWY QQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQG TKLEIKR (SEQ ID NO:487).
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: QVQLVQSGAEVKKPGSSVKVSCKASGGT FSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSE DTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO:488).
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: DIQMTQSPSSLSASVGDR VTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO:487); and a heavy chain comprising the amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMG ATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNW GQGTLVTVSS (SEQ ID NO:488).
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.
(f)Anti-MUC1
In some cases, a targeting sequence present in a MAPP of the present disclosure is an antibody specific for MUC1. For example, a targeting sequence can be specific for a MUC1 polypeptide present on a cancer cell. In some cases, the targeting sequence 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 targeting sequence 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 targeting sequence 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 targeting sequence is a scFv specific for the MUC1 peptide VTSAPDTRPAPGSTAPPAHG (SEQ ID NO:489). In some cases, a targeting sequence is a scFv specific for the MUC1 peptide:
As an example, an anti-MUC1 antibody can comprise: a VH CDR1 having the amino acid sequence RYGMS (SEQ ID NO:494); a VH CDR2 having the amino acid sequence TISGGGTYIYYPDSVKG (SEQ ID NO:495); a VH CDR3 having the amino acid sequence DNYGRNYDYGMDY (SEQ ID NO:496); a VL CDR1 having the amino acid sequence SATSSVSYIH (SEQ ID NO:497); a VL CDR2 having the amino acid sequence STSNLAS (SEQ ID NO:498); and a VL CDR3 having the amino acid sequence QQRSSSPFT (SEQ ID NO:499). See, e.g., US PAT PUB. NO. 2018/0112007.
As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO:500); a VH CDR2 having the amino acid sequence TISSGGTYIYYPD SVKG (SEQ ID NO:501); a VH CDR3 having the amino acid sequence LGGDNYYEYFDV (SEQ ID NO:502); a VL CDR1 having the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO:503); a VL CDR2 having the amino acid sequence LASNLES (SEQ ID NO:504); and a VL CDR3 having the amino acid sequence QHSRELPFT (SEQ ID NO:505). See, e.g., US 2018/0112007.
As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence DYAMN (SEQ ID NO:506); a VH CDR2 having the amino acid sequence VISTFSGNINFN QKFKG (SEQ ID NO:507); a VH CDR3 having the amino acid sequence SDYYGPYFDY (SEQ ID NO:508); a VL CDR1 having the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO:509); a VL CDR2 having the amino acid sequence KVSNRFS (SEQ ID NO:510); and a VL CDR3 having the amino acid sequence FQGSHVPFT (SEQ ID NO:511). 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:500); a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO:512); a VH CDR3 having the amino acid sequence LGGDNYYEY (SEQ ID NO:513); a VL CDR1 having the amino acid sequence TASKSVSTSGYSYMH (SEQ ID NO:514); a VL CDR2 having the amino acid sequence LVSNLES (SEQ ID NO:515); and a VL CDR3 having the amino acid sequence QHIRELTRSE (SEQ ID NO:516). See, e.g., US 2018/0112007.
(g)Anti-MUC16
In some cases, a targeting sequence present in a MAPP of the present disclosure 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 targeting sequence 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 targeting sequence is a scFv. In some cases, a MUC16-specific targeting sequence is a nanobody.
As one example, an anti-MUC16 antibody can comprise a VH CDR1 having the amino acid sequence GFTFSNYY (SEQ ID NO:517); a VH CDR2 having the amino acid sequence ISGRGSTI (SEQ ID NO:518); a VH CDR3 having the amino acid sequence VKDRGGYSPY (SEQ ID NO:519); a VL CDR1 having the amino acid sequence QSISTY (SEQ ID NO:520); a VL CDR2 having the amino acid sequence TAS; and a VL CDR3 having the amino acid sequence QQSYSTPPIT (SEQ ID NO:521). See, e.g., US 2018/0118848.
(h)Anti-Claudin-18.2
In some cases, a targeting sequence present in a MAPP of the present disclosure 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 targeting sequence is a scFv. In some cases, a claudin-18.2-specific targeting sequence is a nanobody. In some cases, a CTP present in a MAPP of the present disclosure is an antibody specific for TEDEVQSYPSKHDYV (SEQ ID NO:449) or EVQSYPSKHDYV (SEQ ID NO:450).
As one example, an anti-claudin-18.2 antibody can comprise a VH CDR1 having the amino acid sequence GYTFTDYS (SEQ ID NO:522); a VH CDR2 having the amino acid sequence INTETGVP (SEQ ID NO:523); a VH CDR3 having the amino acid sequence ARRTGFDY (SEQ ID NO:524); a VL CDR1 having the amino acid sequence KNLLHSDGITY (SEQ ID NO:525); a VL CDR2 having the amino acid sequence RVS; and a VL CDR3 having the amino acid sequence VQVLELPFT (SEQ ID NO:526).
As another example, an anti-claudin-s antibody can comprise a VH CDR1 having the amino acid sequence GFTFSSYA (SEQ ID NO:527); a VH CDR2 having the amino acid sequence ISDGGSYS (SEQ ID NO:528); a VH CDR3 having the amino acid sequence ARDSYYDNSYVRDY (SEQ ID NO:529); a VL CDR1 having the amino acid sequence QDINTF (SEQ ID NO:530); a VL CDR2 having the amino acid sequence RTN; and a VL CDR3 having the amino acid sequence LQYDEFPLT (SEQ ID NO:531).
(iii) Single-Chain T Cell Receptors
In some cases, a CTP present in a MAPP of the present disclosure is a scTCR. A CTP 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 fragment 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 Vβ through a suitable peptide linker (L) sequence fused to the C-terminus of the Vα and the N-terminus of the Vβ. A scTCR can have the structure Vα-Vβ. A scTCR can have the structure Vβ-L-Vα. A scTCR can also comprise a constant domain (also referred to as constant region). In some cases, a 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, a 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:545) peptide bound to an HLA complex comprising an HLA-A*0201 heavy chain and a β2M polypeptide. As an example, such a scTCR can comprise: i) a TCR α chain variable region comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNL QWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGG SYIPTFGRGTSLIVHPY (SEQ ID NO:394), where aa 20 can be V or A; aa 51 can be Q, P, S, T, or M; aa 52 can be S, P, F, or G, aa 53 can be S, W, H, or T; aa 94 can be P, H, or A; aa 95 can be T, L, M, A, Q, Y, E, I, F, V, N, G, S, D, or R; aa 96 can be S, L, T, Y, I, Q, V, E, A, W, R, G, H, D, or K; aa 97 can be G, D, N, V, S, T, or A; aa 98 can be G, P, H, S, T, W, or A; aa 99 can be S, T, Y, D, H, V, N, E, G, Q, K, A, I, or R; aa 100 can be Y, F, M, or D; aa 101 can be I, P, T, or M; and aa 103 can be T or A; and ii) a TCR β chain variable region comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: MGVTQTPKFQVLKT GQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLR LLSAAPSQTSVYFCASSYVGNTGELFFGEGSR LTVL (SEQ ID NO:395), where aa 18 can be M or V; aa 50 can be G, V, or I; aa 52 can be G or Q; aa 53 can be I, T, or M; aa 55 can be D or R; aa 56 can be Q or R; aa 70 can be T or I; aa 94 can be Y, N, or F; aa 95 can be V or L; and aa 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 aa sequence: MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIMSHQR EQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIV HPY (SEQ ID NO:396); and a TCR β chain variable region comprising the aa sequence: MGVTQTPKFQVLKT GQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVSAGITDQGEVPNGYNVSRSTTEDFPLR LLSAAPSQTSVYFCASSYVGNTGELFFGEGSR LTVL (SEQ ID NO:397).
As another example, a scTCR can be specific for an HPV peptide epitope (e.g., an HPV peptide of the aa sequence YIIFVYIPL (HPV 16 E563-71; SEQ ID NO:398), KLPQLCTEL (HPV 16 E611-19; SEQ ID NO:399), TIHEIILECV (HPV 16 E6; SEQ ID NO:400), YMLDLQPET (HPV 16 E711-19; SEQ ID NO:401), TLGIVCPI (HPV 16 E786-93; SEQ ID NO:252), KCIDFYSRI (HPV 18 E667-75; SEQ ID NO:402), or FQQLFLNTL (HPV 18 E786-94; SEQ ID NO:403)) bound to an HLA complex comprising an HL heavy chain and a β2M polypeptide. As an example, such a scTCR can comprise: i) a TCR α chain variable region comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence: METLLGLLILQLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT SLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRETSGSRLTFGEGTQLTV NPD (SEQID NO:404); and ii) a TCR β chain variable region comprising an aa sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, aa sequence identity to the aa sequence:
A polypeptide chain of a MAPP can comprise a payload such as a therapeutic (e.g., a small molecule drug or therapeutic) a label (e.g., a fluorescent label or radio label), or other biologically active agent that is linked (e.g., covalently attached) to the polypeptide chain. For example, where a MAPP comprises an Fc polypeptide, the Fc polypeptide may comprise a covalently linked payload such as an agent that treats a cancer, infectious disease, or an autoimmune disease, potentates the action of the MAPP, or is an agent that relieves a symptom of such diseases.
A payload can be linked directly or indirectly to a polypeptide chain of a MAPP (e.g., to an Ig Fc polypeptide in the MAPP). Direct linkage can involve linkage to an aa side chain without an intervening linker. Indirect linkage can be linkage via a cross-linker, such as a bifunctional cross-linker. A payload can be linked to a MAPP by any acceptable chemical linkage including, but not limited to a thioether bond, an amide bond, a carbamate bond, a disulfide bond, or an ether bond, including those formed by reaction with a crosslinking agent.
Crosslinkers (crosslinking agents) include cleavable crosslinkers and non-cleavable cross-linkers. The cross-linkers may be homobifunctional or heterobifunctional cross-linkers. In some cases, the cross-linker is a protease-cleavable cross-linker. Suitable cross-linkers may include as moieties, for example, peptides (e.g., from 2 to 10 aas in length; e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 aas in length), alkyl chains, poly(ethylene glycol), disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Non-limiting example of suitable cross-linkers are: N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]estr (NHS-PEG4-maleimide); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB); N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB); N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP); N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC); κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA); γ-maleimide butyric acid N-succinimidyl ester (GMBS); ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS); m-maleimide benzoyl-N-hydroxysuccinimide ester (MBS); N-(α-maleimidoacetoxy)-succinimide ester (AMAS); succinimidyl-6-(β-maleimidopropionamide)hexanoate (SMPH); N-succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); N-(p-maleimidophenyl)isocyanate (PMPI); N-succinimidyl 4(2-pyridylthio)pentanoate (SPP); N-succinimidyl(4-iodo-acetyl)aminobenzoate (SIAB); 6-maleimidocaproyl (MC); maleimidopropanoyl (MP); p-aminobenzyloxycarbonyl (PAB); N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC); N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), a “long chain” analog of SMCC (LC-SMCC); 3-maleimidopropanoic acid N-succinimidyl ester (BMPS); N-succinimidyl iodoacetate (SIA); N-succinimidyl bromoacetate (SBA); and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).
MAPP payload conjugates may be formed by reaction of a MAPP polypeptide (e.g., an IgFc polypeptide) with a cross-linking reagent to introduce 1-10 reactive groups. The polypeptide is then reacted with the molecule to be conjugated (e.g., a thiol-containing payload drug, label or agent) to produce a MAPP-payload conjugate. For example, where a MAPP comprises an IgFc polypeptide, the conjugate can be of the form (A)-(L)-(C), where (A) is the polypeptide chain comprising the IgFc polypeptide; where (L), if present, is a cross-linker; and where (C) is a payload. (L), if present, links (A) to (C). In some cases, the MAPP includes an IgFc polypeptide that comprises one or more (e.g., 2, 3, 4, 5, or more than 5) molecules of a payload. Introducing payloads into a MAPP using an excess of cross-linking agents can result in multiple molecules of payload being incorporated into the MAPP.
Suitable payloads (e.g., drugs) include virtually any small molecule (e.g., less than 2,000 Daltons in molecular weight) approved by the U.S. Food and Drug Administration, and/or listed in the 2020 U.S. Pharmacopeia or National Formulary. In an embodiment, those drugs are less than 1,000 molecular weight. Suitable drugs include antibiotics, chemotherapeutic (antineoplastic), anti-fungal, or anti-helminth agents and the like (e.g., sulfasalazine, azathioprine, cyclophosphamide, leflunomide; methotrexate, antimalarials, D-penicillamine, cyclosporine). Suitable chemotherapeutics may be alkylating agents, cytoskeletal disruptors (taxanes), epothilone, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analog or precursor analogs, peptide antineoplastic antibiotics (e.g., bleomycin or actinomycin), platinum-based agents, retinoids, or vinca alkaloids. Suitable drugs also include non-steroidal anti-inflammatory drugs and glucocorticoids, and the like.
D. Nucleic Acids
The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding one or more polypeptides of a MAPP of the present disclosure. In some cases, the nucleic acid is a recombinant expression vector; thus, the present disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a MAPP of the present disclosure.
The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a MAPP having a framework polypeptide that comprises at least one dimerization sequence and at least one multimerization sequence that permits two molecules of the framework polypeptide to form dimers or higher order complexes. The nucleic acids may additionally comprise a nucleotide sequence encoding a dimerization peptide. Where the MAPP comprises a presenting sequence the nucleic acids encoding either or both of the framework polypeptide and/or dimerization peptide may include a sequence encoding a presenting sequence. Where the MAPP comprises a presenting complex, the nucleic acids encoding either or both of the framework polypeptide and/or dimerization peptide may further comprise sequences encoding a presenting complex 1st sequence and/or a presenting complex 2nd sequence. Where desired, the nucleic acid sequences encoding a MAPP may also exclude a sequence encoding a peptide epitope. The nucleotide sequence(s) comprising any of the MAPP polypeptides can be operably linked to a transcription control element(s), e.g., a promoter. It will be apparent that individual polypeptides of a MAPP (e.g., a framework polypeptide and dimerization polypeptide) may be encoded on a single nucleic acid (e.g., under the control of separate promoters), or alternatively, may be located on two or more separate nucleic acids (e.g., plasmids).
The present disclosure provides recombinant expression vectors comprising nucleic acids encoding one or more polypeptides of a MAPP or its higher order complexes. In some cases, the recombinant expression vector is a non-viral vector. In some cases, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, a non-integrating viral vector, etc.
Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Virol. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available.
Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see, e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).
In some cases, a nucleotide sequence encoding one or more polypeptides of a MAPP is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. The transcriptional control element may be functional in either a eukaryotic cell, e.g., a mammalian cell such as a human, hamster, or mouse cell; or a prokaryotic cell (e.g., bacterial). In some cases, a nucleotide sequence encoding a DNA-targeting RNA and/or a site-directed modifying polypeptide is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding a DNA-targeting RNA and/or a site-directed modifying polypeptide in both prokaryotic and eukaryotic cells.
Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include the cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression.
E. Genetically Modified Host Cells
The present disclosure provides a genetically modified host cell, where the host cell is genetically modified with a nucleic acid(s) of the present disclosure that encode, or encode and express, MAPP proteins or higher order complexes of MAPPs (e.g., duplex MAPPs).
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. CRL-9618™, CCL-61™, 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. CCL-10™), PC12 cells (ATCC No. CRL-1721™), 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 β2M and/or such that it does not synthesize endogenous MHC Class I heavy chains (MHC-H).
Genetically modified host cells can be used to produce a MAPP and higher order complexes of MAPPs. For example, a genetically modified host cell can be used to produce a duplex MAPP. For example, an expression vector(s) comprising nucleotide sequences encoding the MAPP polypeptide(s) is/are introduced into a host cell, generating a genetically modified host cell, which genetically modified host cell produces the polypeptide(s) (e.g., as an excreted soluble protein).
F. Methods of Producing MAPPs
The present disclosure provides methods of producing the MAPPs (e.g., duplex MAPPs) described herein. The methods generally involve culturing, in a culture medium, a host cell that is genetically modified with a recombinant expression vector(s) comprising a nucleotide sequence(s) encoding the MAPP (e.g., a genetically modified host cell of the present disclosure); and isolating the MAPP from the genetically modified host cell and/or the culture medium. As noted above, in some cases, the individual polypeptide chains of a MAPP are encoded in separate nucleic acids (e.g., recombinant expression vectors). In some cases, all polypeptide chains of a MAPP are encoded in a single recombinant expression vector.
Isolation of the MAPP from the host cell employed for expression (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 of the host cell may be prepared, and the MAPP purified from the lysate using high performance liquid chromatography (HPLC), exclusion chromatography (e.g., size exclusion chromatography), gel electrophoresis, affinity chromatography, or other purification technique. Alternatively, where the MAPP is secreted from the expression host cell into the culture medium, the MAPP can be purified from the culture medium using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some cases, the MAPP is purified, e.g., a composition is generated that comprises at least 80% by weight, at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, of the MAPP 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 expressed MAPP comprises an affinity tag or affinity domain, the MAPP can be purified using an immobilized binding partner of the affinity tag. For example, where a MAPP comprises an Ig Fc polypeptide, the MAPP can be isolated from genetically modified mammalian host cell and/or from culture medium comprising the MAPP by affinity chromatography, e.g., on a Protein A column, a Protein G column, or the like. An example of a suitable mammalian cell is a CHO cell; e.g., an Expi-CHO-S™ cell (e.g., ThermoFisher Scientific, Catalog #A29127).
The polypeptides of the MAPP will self-assemble into heterodimers, and where applicable, spontaneously form disulfide bonds between, for example, framework polypeptides, or framework and dimerization polypeptides. As also noted above, when both framework polypeptides include Ig Fc polypeptides, disulfide bonds will spontaneously form between the respective Ig Fc polypeptides to covalently link the two heterodimers of framework and dimerization polypeptides to one another to form a covalently linked duplex MAPP.
G. Compositions
1. Compositions Comprising a MAPP
The present disclosure provides compositions, including pharmaceutical compositions, comprising a MAPP and/or higher order complexes of MAPPs (e.g., duplex MAPPs). Pharmaceutical composition can comprise, in addition to a MAPP , one or more known carriers, excipients, diluents, buffers, salts, surfactants (e.g., non-ionic surfactants), amino acids (e.g., arginine), etc., a variety of which are known in the art and need not be discussed in detail herein. For example, see “Remington: The Science and Practice of Pharmacy”, 19th Ed. (1995), or latest edition, Mack Publishing Co.
In some cases, a subject pharmaceutical composition will be suitable for administration to a subject, e.g., will be sterile and/or substantially free of pyrogens. For example, in some embodiments, a subject pharmaceutical composition will be suitable for administration to a human subject, e.g., where the composition is sterile and is substantially free of detectable pyrogens and/or other toxins, or such detectable pyrogens and/or other toxins are below a permissible limit.
The compositions may, for example, be in the form of aqueous or other solutions, powders, granules, tablets, pills, suppositories, capsules, suspensions, sprays, and the like. The composition may be formulated according to the various routes of administration described below.
Where a MAPP or higher order MAPP complex (e.g., duplex MAPP) is administered as an injectable (e.g., subcutaneously, intraperitoneally, intramuscularly, intralymphatically, and/or intravenously) directly into a tissue, a formulation can be provided as a ready-to-use dosage form, or as non-aqueous form (e.g., a reconstitutable storage-stable powder) or an aqueous form, such as liquid composed of pharmaceutically acceptable carriers and excipients. MAPPs may also be provided so as to enhance serum half-life of the subject protein following administration. For example, the protein may be provided in a liposome formulation, prepared as a colloid, or other conventional techniques for extending serum half-life. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The preparations may also be provided in controlled release or slow-release forms.
In some cases, a MAPP composition comprises: a) a MAPP higher order MAPP complex (e.g., a duplex MAPP) of the present disclosure; and b) saline (e.g., 0.9% NaCl). In some cases, the composition is sterile and/or substantially pyrogen free. In some cases, the composition is suitable for administration to a human subject, e.g., where the composition is sterile and is free of detectable pyrogens and/or other toxins. Thus, the present disclosure provides a composition comprising: a) a MAPP or higher order MAPP complex (e.g., duplex MAPP) of the present disclosure; and b) saline (e.g., 0.9% NaCl), where the composition is sterile and is substantially free of detectable pyrogens and/or other toxins, or such detectable pyrogens and/or other toxins are below a permissible limit.
Other examples of components suitable for inclusion in formulations suitable for parenteral administration include isotonic sterile injection solutions, anti-oxidants, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. A pharmaceutical composition can be present in a container, e.g., a sterile container, such as a syringe. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
The concentration of a MAPP in a formulation can vary widely. For example, a MAPP or higher order MAPP complex (e.g., duplex MAPP) may be present from less than about 0.1% (usually at least about 2%) to as much as 20% to 50% or more by weight (e.g., from 1% to 10%, 5% to 15%, 10% to 20% by weight, or 20-50% by weight) by weight. The concentration will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected and the patient's needs.
The present disclosure provides a container comprising a composition of the present disclosure, e.g., a liquid composition. The container can be, e.g., a syringe, an ampoule, and the like. In some cases, the container is sterile. In some cases, both the container and the composition are sterile.
The present disclosure provides compositions (e.g., pharmaceutical compositions) comprising a nucleic acid or a recombinant expression vector that comprise one or more nucleic acid sequences encoding any one or more MAPP polypeptides (or each of the polypeptides of a MAPP). As discussed above, a wide variety of pharmaceutically acceptable excipients is known in the art and need not be discussed in detail herein.
A nucleic acid or a recombinant expression vector composition can include one or more nucleic acids or one or more recombinant expression vectors comprising a nucleic acid (e.g., DNA or RNA) sequences encoding a MAPP polypeptide or all polypeptides of a MAPP. Such compositions may further include one or more of: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative.
A pharmaceutically acceptable formulation may comprise a nucleic acid or recombinant expression vector encoding one or more polypeptides of a MAPP (e.g., in an amount of from about 0.001% to about 90% (w/w)). In some cases, such pharmaceutical compositions will be suitable for administration to a subject, e.g., will be sterile and/or substantially free of pyrogens. For example, in some embodiments, a the pharmaceutical composition will be suitable for administration to a human subject, e.g., where the composition is sterile and is substantially free of detectable pyrogens and/or other toxins, or such detectable pyrogens and/or other toxins are below a permissible limit.
A composition comprising a nucleic acid or a recombinant expression vector encoding one or more polypeptides of a MAPP, including pharmaceutically acceptable formulations, may be:
The compositions comprising a nucleic acid or a recombinant expression vector described herein may include penetration enhancers to effect the efficient delivery of nucleic acids or expression vectors. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in, for example, U.S. Pat. No. 6,287,860, which is incorporated for its discussion penetration enhancers.
H. Methods
MAPPs and higher order MAPP complexes (e.g., duplex MAPP) of the present disclosure are useful for modulating an activity of a T cell. Thus, the present disclosure provides methods of modulating an activity of a T cell, the methods generally involving contacting a target T cell with a MAPP or a higher order MAPP complex (e.g., duplex MAPP) of the present disclosure.
The present disclosure provides a method of selectively modulating the activity of a T cell that is specific for an epitope presented by a MAPP, the method comprising contacting, in vitro or in vivo, the T cell with a MAPP, where contacting the T cell with a MAPP or higher order MAPP, such as a duplex MAPP, presenting the epitope selectively modulates the activity of the epitope-specific T cell. The contacting may occur in vivo, typically in a human, but potentially in another animal such as a rat, mouse, dog, cat, pig, horse, or primate. In some cases, the contacting occurs in vitro.
In some cases, contacting a MAPP with a T cell that is specific for the epitope presented by the MAPP result in one or more of: i) activating or suppression of a cytotoxic (e.g., CD8+) T cell; ii) inducing or suppression of a cytotoxic activity of a cytotoxic (e.g., CD8+) T cell; iii) inducing or suppressing the production and release of one or more cytotoxic molecules (e.g., a perforin; a granzyme; a granulysin) by a cytotoxic (e.g., CD8+) T cell; iv) inhibiting activity of an autoreactive T cell; v) induction of CD8+ T regulatory cells; and the like.
In some cases, a MAPP or higher order MAPP complex (e.g., duplex MAPP) of the present disclosure interacts with an epitope specific CD 8+ T cell and increases granule-dependent and/or granule-independent CD8+ effector T cell responses. Granule-independent responses include, but are not limited to, changes in the number or percentage of active epitope-specific CD 8+ T cell (e.g., in a population of cells such as in blood, lymphatics, and/or in a target tissue), changes in the expression of Fas ligand (Fas-L, which can result in activation of caspases and target cell death through apoptosis), and cytokine/chemokine production (e.g., production and release of interferon gamma (IFN-γ). Granule-dependent effector actions include the release of granzymes, perforin, and/or granulysin. Activation of epitope specific CD8+ cytotoxic T cells (e.g., CD8+ cytotoxic effector T cells) can result in the targeted killing of, for example, cancer cells and/or infected cell by epitope specific T cells that recognize the epitope presented by the MAPP or MAPP complex through granule-dependent and/or independent responses.
In some cases a MAPP or higher order MAPP complex of the present disclosure comprises a cancer epitope, and the MAPP or MAPP complex activates a CD8+ T cell response (e.g., a CD8+ T cell response to a cancer cell). In some cases, a MAPP or higher order MAPP complex of the present disclosure comprises an epitope of an infectious agent, and it activates a CD8+ T cell response (e.g., a CD8+ T cell response to a cell expressing an antigen of an infectious agent).
The present disclosure provides a method of increasing proliferation and/or number of CD 8+ effector T cells specific to the epitope presented by a MAPP or MAPP complex, the method comprising contacting (e.g., in vitro or in vivo) T cells with a MAPP or higher order MAPP complex (such as by administering to a subject one or more doses of a epitope-presenting MAPP or MAPP complex with a MOD (e.g., IL-2)). The contacting or administering may increase the number of CD8+ effector T cells having a TCR capable of binding the epitope present in the MAPP or MAPP complex relative to the number (e.g., total number or percentage) of T cells present (e.g., in a population of cells such as in blood, lymphatics, and/or in a target tissue). For example, the number of CD 8+ effector T cells specific to the epitope presented by the MAPP or MAPP complex (e.g., duplex MAPP) can be increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, least 75%, at least 100%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold following one or more contacts with doses or administrations of the MAPP or higher order MAPP complex. The increase may be calculated relative the CD+ T cell numbers present prior to the contacting or administrations, or relative to the population of T cells present in a sample (e.g., a sample of blood or tissue) that has not been contacted with the MAPP or MAPP complex.
The present disclosure provides a method of increasing granule-dependent and/or granule-independent responses of epitope-specific CD 8+ T cell comprising contacting (e.g., in vitro or in vivo) T cells with a epitope presenting MAPP or higher order MAPP complex (such as by administering to a subject one or more does of a MAPP or MAPP complex with, for example a CD80 or CD86 MOD). The contacting or administering results in increasing the expression of Fas ligand expression, cytokines/chemokines (e.g., IL-2, IL-4, and/or IL-5), release of interferons (e.g., IFN-γ), release of granzymes, release of perforin, and/or release of granulysin. For example, contacting CD 8+ effector cell with a MAPP or MAPP complex (e.g., duplex MAPP) presenting epitope specific to the effector cell can increase one or more of Fas ligand expression, interferon gamma (IFN-γ) release, granzyme release, perforin release, and/or granulysin release by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, least 75%, at least 100%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold. The increase may be calculated relative the level of expression or release prior to the contacting or administrations, or relative to the population of T cells present in a sample (e.g., a sample of blood or tissue) that has not been contacted with the MAPP or MAPP complex.
Dysregulation of CD8+ T reg cells and self-reactive CD8+ effector T cells have both been associated with the pathogenesis of autoimmune diseases including, but not limited to, multiple sclerosis, Rasmussen's encephalitis, paraneoplastic syndromes, systemic sclerosis (SSc), Grave's disease (GD), systemic lupus erythematosus (SLE), aplastic anemia (AA), and vitiligo (see e.g., Pilli et al, Frontiers in Immunology, Article 652, vol. 8, June 2017; Coppieters et al, J. Exp. Med. Vol. 209 No. 1 51-60 (2012); Han et al., PNAS (USA), 110(32):13074-13078 (2013) and Pellegrino et al. PLOS ONE, https://doi.org/10.1371/journal.pone.0210839 January 16, (2019). Deng et al, has reviewed the epigenetic role of CD8+ T cell in autoimmune diseases (see Deng et al, Frontiers in Immunology, Article 856, vol. 10, April 2019).
CD8+ effectors may promote autoimmune diseases via dysregulated secretion of inflammatory cytokines, skewed differentiation profiles and inappropriate apoptosis or induction of effector T cells directed against target cells. In some cases, a MAPP or a higher order MAPP complex (e.g., duplex MAPP) of the present disclosure presenting an auto-antigen (self-antigen or self-epitope) may reduce the activity of an autoreactive CD8+ effector T cells by direct interaction with the cell. Contacting a MAPP or higher order MAPP complex (e.g., duplex MAPP) of the present disclosure presenting an autoantigen and one or more independently selected inhibitory MODs (e.g., PDL1, or FasL) with an autoreactive CD8+ effector T cell may be employed to block autoimmune disease by regulating (e.g., reducing) the release of proinflammatory molecules by such T cells and/or by eliminating self-reactive cells.
Where it is desirable to reduce the activity of epitope specific T cells (e.g., where they are directed against an autoantigen) they may be contacting MAPPs or MAPP complexes (e.g., duplex MAPP) presenting the epitope and bearing MODs that modulate their epitope specific response. Modulation of the cytotoxic CD8+ T cells by MAPP and MAPP complexes may result in, but is not limited to, one or more of: i) suppression of FasL expression by the T cell; ii) suppression of chemokine and/or cytokine release (e.g., IFN-γ); and/or iii) suppression of cytotoxin (e.g., a perforin; a granzyme; a granulysin) synthesis or release. The disclosure includes and provides for a method of reducing (e.g., in vivo, or in vitro) effector T cell activity in an epitope specific manner, such as where the T cell is directed to an autoantigen. The method comprises contacting (such as by administering to a subject) an epitope-specific T cell with one or more doses of a MAPP or MAPP complex presenting the epitope and bearing a MOD (e.g., TGF-β); with the contacting or administering resulting in a reduction in one or more of: i) FasL expression by the T cell; ii) suppression of chemokine and/or cytokine release (e.g., IFN-γ); and iii) suppression of cytotoxin (e.g., a perforin; a granzyme; a granulysin) synthesis or release by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, least 75%, at least 100%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold. The change may be calculated relative the level of expression or release prior to the contacting or administrations, or relative to the population of T cells present in a sample (e.g., a sample of blood or tissue) that has not been contacted with the MAPP or MAPP complex.
In other instances, a MAPP or a higher order MAPP complex presenting and antigen (e.g., an autoantigen) may interact with and increase the number or activity of CD8+ regulatory T cells (CD8+ T regs, characterized e.g., as CD8+ FOXP3+ or CD8+ FOXP3+ CD25+) specific to the antigen. Various CD8+ Treg subsets function by, for example, secreting cytokines and chemokines, including IL-10, TGF-β, IL-16, IFN-γ and chemokine (C-C motif) ligand 4 (CCL4), and thereby suppressing the activity of effector T cells and potentially the activity of CD4+ T cells subject to those cytokines. CD8+ Tregs may also inhibit T cell function through cell-to-cell contact in which surface proteins such as TGF-β and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) act on the T effector cell. See e.g., Yu et al. Oncol. Lett 15(6):8187-8194 (2018).
The present disclosure provides methods of increasing the number (proliferation) of epitope specific CD8+ Tregs directed to a self-antigen and/or the release of one or more of IL-10, TGF-β, IL-16, IFN-γ and CCL4 and thereby suppressing immune/autoimmune responses. One method of increasing the number of self-antigen (self-epitope) specific CD8+ Tregs (e.g., in a subject) comprises contacting (in vitro or in vivo such as by administering to a subject) an epitope-specific T cell with one or more doses of a MAPP or MAPP complex presenting the self-antigen (self-epitope) and bearing a MOD stimulatory to CD8+ T reg proliferation, where the contacting increases proliferation of CD8+ Tregs by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, least 75%, at least 100%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold relative to the number of CD8+ T regs present in a sample (e.g., a sample of blood or tissue) that has not been contacted with the MAPP or MAPP complex.
The present disclosure also provides methods of increasing one or more of IL-10, TGF-β, IL-16, IFN-γ and CCL4 and thereby suppressing immune/autoimmune responses. The method comprising contacting (in vitro or in vivo such as by administering to a subject) an epitope-specific T cell with one or more doses of a MAPP or MAPP complex presenting the antigen and bearing a MOD stimulatory to CD8+ T reg proliferation. The contacting increases the release at least one of IL-10, TGF-β, IL-16, IFN-γ and CCL4 by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, least 75%, at least 100%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold relative to the amounts prior to the contacting or relative to the population of T cells present in a sample (e.g., a sample of blood or tissue) that has not been contacted with the MAPP or MAPP complex.
Where it is desirable to reduce or substantially eliminate the number of epitope specific CD8+ effectors (e.g., where they are directed against an autoantigen) they may be contacted with a MAPP or MAPP complexes (e.g., duplex MAPP) presenting the epitope and comprising one or more MODs that lead to suppression of the CD8+ T cells (e.g., PDL-1) or apoptosis and/or comprise an Ig Fc region facilitating antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). Apoptosis may occur, for example, when the MAPP or MAPP complex comprises both an epitope (e.g., an autoantigen) and a MOD such as FasL that induces FAS mediated apoptosis. Elimination of epitope specific T cells may also occur as a result of antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) where the MAPP or MAPP complex presents an epitope and comprises an immunoglobulin Fc polypeptide with wt. or enhanced ADCC and/or CDC functionality. Accordingly, the disclosure includes and provides for a method of reducing or substantially eliminating (e.g., in vivo or in vitro) the number of effector T cells in an epitope specific manner, such as where the T cell is directed to an autoantigen. The method comprises contacting (such as by administering to a subject in vivo, or to a cell in vitro) an epitope-specific T cell with one or more doses of a MAPP or MAPP complex presenting the epitope and bearing a MOD resulting in T cell suppression and/or apoptosis. The contacting or administering resulting elimination of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the CD8+ cells specific to the epitope presented by the MAPP or MAPP complex. The change may be calculated relative the number of T cells present in a sample (e.g., a sample of blood or tissue) prior to the contacting (administration) of the MAPP or MAPP complex, or a sample that has not been contacted with the MAPP or MAPP complex.
The present disclosure provides a method of detecting an antigen-specific T-cell. The methods comprise contacting a T cell with a MAPP either lacking a MOD or bearing a MOD with minimal affinity for its co-MOD; and detecting binding of the MAPP to the T cell. Binding of such a MAPP to the T cell indicates that the T cell is specific for the epitope present in the MAPP. In some cases, the MAPP comprises a detectable label. Suitable detectable labels include, but are not limited to, a radioisotope, a fluorescent polypeptide, or an enzyme that generates a fluorescent product, and an enzyme that generates a colored product. Where the MAPP comprises a detectable label, binding of the MAPP to the T cell is detected by detecting the detectable label.
In some cases, an MAPP comprises a detectable label suitable for use in in vivo imaging, e.g., suitable for use in positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging, x-ray imaging, computer-assisted tomography (CAT), or magnetic resonance imaging (MRI), or other in vivo imaging method. Examples of suitable labels for in vivo imaging include gadolinium chelates (e.g., gadolinium chelates with DTPA (diethylenetriamine penta-acetic acid), DTPA-bismethylamide (BMA), DOTA (dodecane tetraacetic acid), or HP-DO3A (1,4,7-tris(carboxymethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazacyclododecane)), iron chelates, magnesium chelates, manganese chelates, copper chelates, chromium chelates, iodine-based materials, and radionuclides.
Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), and the like. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.
Suitable enzymes that may be employed as labels include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, glucose oxidase (GO), and the like.
In some cases, binding of the MAPP to the T cell is detected using a detectably labeled antibody specific for the MAPP. An antibody specific for the MAPP can comprise a detectable label such as a radioisotope, a fluorescent polypeptide, or an enzyme that generates a fluorescent product, or an enzyme that generates a colored product.
In some cases, the T cell being detected is present in a sample comprising a plurality of T cells. For example, a T cell being detected can be present in a sample comprising from 10 to 109 T cells, e.g., from 10 to 102, from 102 to 104, from 104 to 106, from 106 to 107, from 107 to 108, or from 108 to 109, or more than 109, T cells.
The present disclosure provides treatment methods, the methods comprising administering to the individual a composition comprising an amount of a MAPP or higher order MAPP complex (e.g., duplex MAPP) of the present disclosure, or one or more nucleic acids or expression vectors encoding a MAPP that may assemble into a higher order complex (e.g., duplex MAPP), effective to selectively modulate the activity of an epitope-specific T cell in an individual and to treat the individual. In some cases, a treatment method comprises administering to an individual in need thereof a composition comprising one or more recombinant expression vectors comprising nucleotide sequences encoding a MAPP (e.g., a MAPP that may assemble into a higher order MAPP complex) of the present disclosure. In some cases, a treatment method comprises administering to an individual in need thereof one or more mRNA molecules comprising nucleotide sequences encoding a MAPP of the present disclosure. In some cases, a treatment method comprises administering to an individual in need thereof a MAPP or higher order MAPP complex (e.g., duplex MAPP) of the present disclosure. The conditions that can be treated include autoimmune disorders.
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 MAPP or higher order MAPP complex (e.g., duplex MAPP) of the present disclosure, or one or more nucleic acids (e.g., expression vectors; mRNA; etc.) comprising nucleotide sequences encoding the MAPP which may assemble into a higher order complex, where the MAPP or its complex 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 MAPP (e.g., duplex MAPP) of the present disclosure) sufficient to effect treatment of a disease or disorder.
In some cases, a MAPP comprises an inhibitory MOD polypeptide sequence, and the MAPP inhibits activity of the epitope-specific T cell (e.g., effector functions or proliferation). In some cases, the epitope is an epitope of an autoantigen (self-epitope), and a MAPP selectively inhibits the activity of a T cell specific for the epitope of the autoantigen.
The present disclosure provides a method of treating an autoimmune disorder in an individual, the method comprising administering to the individual pharmaceutical composition comprising an effective amount of a MAPP or higher order MAPP complex such as a duplex MAPP of the present disclosure, or one or more nucleic acids comprising nucleotide sequences encoding the MAPP (which may assemble into a higher order complex such as an duplex MAPP), wherein the MAPP (e.g., a MAPP comprising an epitope of an autoantigen), and wherein the MAPP or higher order MAPP complex (e.g., duplex MAPP) comprises an inhibitory MOD (e.g., FasL and/or PDL1). In some cases an “effective amount” of a MAPP or higher order MAPP complex is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number self-reactive T cells 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 number of self-reactive T cells in the individual before administration of the MAPP, or in the absence of MAPP administration. In some cases, an “effective amount” of a MAPP is an amount that, when administered in one or more doses to an individual in need thereof, reduces production of Th2 cytokines (e.g., IL-4, IL-5, and IL-13) in the individual or a tissue of the individual. In some cases, an “effective amount” of a MAPP or higher order MAPP complex (e.g., duplex MAPP) of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, ameliorates one or more symptoms associated with an autoimmune disease in the individual. In some instances, the MAPP or higher order MAPP complex (e.g., duplex MAPP) reduces the number of CD8+ self-reactive T cells. In some instances, the MAPP or higher order MAPP complex (e.g., duplex MAPP) increases the number of CD8+ Tregs, which in turn reduces the number of CD8+ self-reactive T effector cells and/or the cytokines or cytotoxic components (e.g., a perforin; a granzyme; a granulysin) released by activated CD8+ effector cells.
In some cases, the MOD is an activating polypeptide, and the MAPP with its associated epitope activates an epitope-specific T cell that recognizes a cancer or pathogen specific antigen (e.g., a viral or bacterial antigen). In some cases, the T cells are cytotoxic T cells (CD8+ cells). In some cases, a MAPP with its associated epitope increases the activity of a CD8+ effector T cell specific for a cancer cell expressing the epitope. Activation of CD8+ T cells can include increasing proliferation of CD8+ T cells and/or inducing or enhancing release of chemokines and/or cytokines by CD8+ T cells.
“In some cases, a MAPP with its associated epitope reduces proliferation and/or activity of a CD8+ regulatory T cell. In some cases, e.g., where a MAPP or MAPP complex of the present disclosure comprises an inhibitory MOD (e.g., PD-L1, FasL, and the like), the MAPP epitope conjugate reduces the proliferation and/or activity of a CD8+ Treg.
As noted above, in some cases, in carrying out a subject treatment method, a MAPP (e.g., duplex MAPP or higher order MAPP complex) is administered to an individual in need thereof, as the polypeptide per se. In other instances, in carrying out a subject treatment method, one or more nucleic acids comprising nucleotide sequences encoding a MAPP is/are administering to an individual in need thereof. Thus, in other instances, one or more nucleic acids of the present disclosure, e.g., one or more recombinant expression vectors of the present disclosure, is/are administered to an individual in need thereof.
A MAPP or higher order MAPP complex (e.g., duplex MAPP), or one or more nucleic acids encoding such molecules may be administered alone or with one or more additional therapeutic agents or drugs. The therapeutic agents may be administered before, during, or subsequent to MAPP or higher order MAPP complex (e.g., duplex MAPP) or nucleic acids encoding such molecules. When the additional therapeutic agents are administered with a composition or formulation comprising a MAPP or higher order MAPP complex (e.g., duplex MAPP) or nucleic acids encoding such molecules, the therapeutic agent may be administered concurrently with the MAPP. Alternatively, the therapeutic agents may be co-administered with the MAPP as part of a formulation or composition comprising the MAPP or higher order MAPP complex (e.g., duplex MAPP).
Suitable therapeutic agents or drugs that may be administered with a MAPP or higher order MAPP complex include virtually any therapeutic agent, including small molecule therapeutics (e.g., less than 2,000 Daltons in molecular weight) approved by the U.S. Food and Drug Administration, and/or listed in the 2020 U.S. Pharmacopeia or National Formulary. In an embodiment, those therapeutic agents or drugs are less than 1,000 molecular weight. Suitable drugs include antibiotics, chemotherapeutic (antineoplastic), anti-fungal, or anti-helminth agents and the like (e.g., sulfasalazine, azathioprine, cyclophosphamide, leflunomide; methotrexate, antimalarials, D-penicillamine, cyclosporine). Suitable chemotherapeutics may be alkylating agents, cytoskeletal disruptors (taxanes), epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analog or precursor analogs, peptide antineoplastic antibiotics (e.g., bleomycin or actinomycin), platinum-based agents, retinoids, or vinca alkaloids. Suitable drugs also include non-steroidal anti-inflammatory drugs and glucocorticoids, and the like.
In an embodiment, a suitable therapeutic agent that may be administered with a MAPP or higher order MAPP complex comprises an anti-TGF-β antibody, such as Metelimumab (CAT192) directed against TGF-β1 and/or Fresolimub directed against TGF-β1 and TGF-β2, or a TGF-β trap (e.g., Cablivi® caplacizumab-yhdp). Such antibodies would, as a generality, not be administered in conjunction with a MAPP or higher order MAPP complex (e.g., a duplexed MAPP) that comprise a sequence to which the antibodies bind such as a TGF-β1 or TGF-β2 MOD.
In an embodiment, a suitable therapeutic agent that may be administered with a MAPP or higher order MAPP complex comprises one or more antibodies directed against: B lymphocyte antigens (e.g., ibritumomab tiuxetan, obinutuzumab, ofatumumab, rituximab to CD20, brentuximab vedotin directed against CD30, and alemtuzumab to CD52); EGFR (e.g., cetuximab, panitumumab, and necitumumab); VEGF (e.g., bevacizumab); VEGFR2 (e.g., ramucirumab); HER2 (e.g., pertuzumab, trastuzumab, and ado-trastuzumab); PD-1 (e.g., nivolumab and pembrolizumab targeting a check point inhibition); RANKL (e.g., denosumab); CTLA-4 (e.g., ipilimumab targeting check point inhibition); IL-6 (e.g., siltuximab); disialoganglioside (GD2), (e.g., dinutuximab) disialoganglioside (GD2); CD38 (e.g., daratumumab); SLAMF7 (Elotuzumab); both EpCAM and CD3 (e.g., catumaxomab); or both CD19 and CD3 (blinatumomab). Such antibodies would, as a generality, not be administered in conjunction with a MAPP or higher order MAPP complex (e.g., a duplexed MAPP) that comprise a sequence to which any of the administered antibodies bind, or which may block the action of a MOD present in the administered MAPP.
In an embodiment, a suitable therapeutic agent that may be administered with a MAPP or higher order MAPP complex comprises an antibiotic, anti-fungal, and/or anti-helminth agent.
In an embodiment, a suitable therapeutic agent that may be administered with a MAPP or higher order MAPP complex comprises one or more chemotherapeutic agents. Such therapeutic agents may be selected from: alkylating agents, cytoskeletal disruptors (taxanes), epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analog or precursor analogs, peptide antineoplastic antibiotics (e.g., bleomycin or actinomycin), platinum-based agents, retinoids, or vinca alkaloids and their derivatives. In an embodiment, the chemotherapeutic agents are selected from actinomycin all-trans retinoic acid, azacytidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, and vindesine.
The present disclosure provides treatment methods, the methods comprising administering to the individual an amount of a MAPP or higher order MAPP complex of the present disclosure, or one or more nucleic acids or expression vectors encoding the MAPP, effective to selectively modulate the activity of an epitope-specific T cell in an individual and to treat the individual. 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 MAPP or higher order MAPP complex of the present disclosure. In some cases, a treatment method comprises administering to an individual in need thereof one or more mRNA molecules comprising nucleotide sequences encoding a MAPP or higher order MAPP complex of the present disclosure. In some cases, a treatment method comprises administering to an individual in need thereof a MAPP or higher order MAPP complex of the present disclosure.
The present disclosure also provides treatment methods, the methods comprising administering to the individual a pharmaceutical composition comprising an effective amount of a MAPP of the present disclosure, or one or more nucleic acids or expression vectors encoding the MAPP optionally bearing an immunoglobulin sequence that can support complement-dependent cytotoxicity (CDC) or antibody-dependent cell cytotoxicity (ADCC). Such MAPPs can selectively engage with an epitope-specific T cell in an individual in order to treat the individual, (e.g., by depleting epitope-specific T cells by inducing cell lysis through activation of CDC, and/or ADCC).
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 MAPP or higher order MAPP complex of the present disclosure, or one or more nucleic acids (e.g., expression vectors; mRNA; etc.) comprising nucleotide sequences encoding the MAPP or higher order MAPP complex, which 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 MAPP or higher order MAPP complex in order to treat a disease or disorder.
In some cases, the MOD is an inhibitory polypeptide, and a MAPP or higher order MAPP complex inhibits activity of the epitope-specific T cell. In some cases, the epitope an epitope of an autoantigen, and a MAPP or higher order MAPP complex selectively inhibits the activity of a T cell specific for the epitope of the autoantigen.
The present disclosure provides a method of treating an autoimmune disorder in an individual, the method comprising administering to the individual an effective amount of a MAPP or higher order MAPP complex that comprises an epitope of an autoantigen and an inhibitory MOD (or one or more nucleic acids comprising nucleotide sequences encoding those molecules. In some cases, an “effective amount” of a MAPP or higher order MAPP complex is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of self-reactive T cells specific to the epitope presented by the MAPP or higher order MAPP complex 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 number of those self-reactive T cells in the individual before or in the absence of administration of the MAPP or higher order MAPP complex. In some cases, an “effective amount” of a MAPP or higher order MAPP complex is an amount that, when administered in one or more doses to an individual in need thereof, reduces production of Th2 cytokines in the individual. In some cases, an “effective amount” of a MAPP or higher order MAPP complex is an amount that, when administered in one or more doses to an individual in need thereof, ameliorates one or more symptoms associated with an autoimmune disease in the individual. In some instances, the MAPP or higher order MAPP complex reduces the number of CD8+ self-reactive T cells specific to the epitope presented by those molecules, which may in turn may lead to a reduction in CD4+ self-reactive T cells. In some instances, a MAPP or higher order MAPP complex increases the number of CD8+ Tregs specific to the epitope presented by those molecules, which in turn reduces the number or activity of CD8+ self-reactive T cells.
In some cases, the MOD is an activating polypeptide, and the MAPP with its associated epitope activates an epitope-specific T cell that recognizes a cancer or pathogen specific antigen (e.g., a viral or bacterial antigen). In some cases, the T cells are cytotoxic T cells (CD8+ cells). In some cases MAPP or higher order MAPP complex with its associated epitope increases the activity of a T cell specific for a cancer cell expressing the epitope (e.g., cytotoxic CD8+ T cells). Activation of CD8+ T cells can include increasing proliferation of CD8+ T cells and/or inducing or enhancing release cytokines by CD8+ T cells such as interferon γ CD8+ cells.
In some cases where a MAPP or higher order MAPP complex comprises an inhibitory MOD (e.g., PD-L1, FasL, and the like) it may reduce the proliferation and/or activity of a CD8+ regulatory T (Treg) cell (e.g., FoxP3+, CD8+ T cells) specific to the epitope presented by the MAPP or higher order MAPP complex.
As noted above, in some cases, in carrying out a subject treatment method, a MAPP or higher order MAPP complex is administered to an individual in need thereof, as the polypeptide per se. In other instances, in carrying out a subject treatment method, one or more nucleic acids comprising nucleotide sequences encoding a MAPP is/are administering to an individual in need thereof. Thus, in other instances, one or more nucleic acids of the present disclosure, e.g., one or more recombinant expression vectors of the present disclosure, is/are administered to an individual in need thereof.
The present disclosure provides a method of delivering a MOD polypeptide such as IL-2, 4-1BBL, CD-80, CD-86, Fas-L, PD-L1, or a reduced-affinity variant of any thereof (e.g., PD-L1 and/or an IL-2 variant disclosed herein) to a selected T cell or a selected T cell population, e.g., in a manner such that a TCR specific for a given epitope is targeted. The present disclosure thus provides a method of delivering a MOD polypeptide such as a IL-2 polypeptide, or a reduced-affinity variant of a naturally occurring MOD polypeptide such as an IL-2 variant, selectively to a target T cell bearing a TCR specific for the peptide epitope sequence present in a MAPP or higher order MAPP complex (e.g., duplex MAPP). The method comprises contacting a population of T cells with a MAPP or higher order MAPP complex (e.g., duplex MAPP). The population of T cells can be a mixed population that comprises: i) the target T cell with a TCR specific to a target epitope; and ii) non-target T cells that are not specific for the target epitope presented by the peptide epitope (e.g., T cells that are specific for an epitope(s) other than the epitope to which the epitope-specific T cell binds). The epitope-specific T cell is specific for the peptide epitope present in the MAPP or higher order MAPP complex, and binds to the peptide MHC complex provided by the MAPP or higher order MAPP complex. Contacting the population of T cells with the MAPP or higher order MAPP complex delivers the MOD polypeptide (e.g., IL-2 or a reduced-affinity variant of IL-2) selectively to the T cell(s) that are specific for the epitope present in the MAPP or higher order complex.
Thus, the present disclosure provides a method of delivering a MOD polypeptide such as PD-L1, or a reduced-affinity variant of a naturally occurring MOD polypeptide such as a PD-L1 variant disclosed herein, or a combination of both, selectively to a target T cell selective for the epitope presented by the peptide epitope as presented by the MAPP. Similarly, the disclosure provides a method of delivering an IL-2, MOD polypeptide or a reduced-affinity variant of a naturally occurring IL-2 MOD polypeptide such as disclosed herein, or a combination of both, to a target T cell that is selective for the epitope presented by the peptide epitope as presented by the MAPP. In some cases, the IL-2 MOD bears a substitution at position H16 and/or F42 (e.g., H16 and F42 such as H16A and F42A) (see supra).
For example, a MAPP (such as a duplex MAPP or higher order MAPP complex) is contacted with a population of T cells comprising: i) target T cells that are specific for the epitope present in the MAPP or a higher order MAPP complex; and ii) non-target T cells, e.g., a T cells that are specific for a second epitope(s) that is not the epitope present in the MAPP or a higher order MAPP complex. Contacting the population results in substantially selective delivery of the MOD polypeptide(s) (e.g., naturally occurring or variant MOD polypeptide) to the target T cell. Less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 4%, 3%, 2% or 1%, of the MAPP or higher order MAPP complex (e.g., duplex MAPP) may bind to non-target T cells and, as a result, the MOD polypeptide (e.g., IL-2 or IL-2 variant) is selectively delivered to target T cell (and accordingly, not effectively delivered to the non-target T cells).
The population of T cells to which a MOD and/or variant MOD is selectively delivered may be in vivo. In some cases, the population of T cells to which a MOD and/or variant MOD is selectively delivered is in vitro.
In some cases, the population of T cells to which a MOD and/or variant MOD is selectively delivered is in vitro. For example, a mixed population of T cells is obtained from an individual, and is contacted with a MAPP (such as a duplex MAPP or higher order MAPP complex) in vitro. Such contacting, which can comprise single or multiple exposures of the T cells to a defined dose(s) and/or exposure schedule(s) in the context of in vitro cell culture, can be used to determine whether the mixed population of T cells includes T cells that are specific for the epitope presented by the MAPP (e.g., duplex MAPP or higher order MAPP complex). The presence of T cells that are specific for the epitope of the MAPP or higher order MAPP complex can be determined by assaying a sample comprising a mixed population of T cells, which population of T cells comprises T cells that are not specific for the epitope (non-target T cells) and may comprise T cells that are specific for the epitope (target T cells). Known assays can be used to detect the desired modulation of the target T cells, thereby providing an in vitro assay that can determine whether a particular MAPP or higher order MAPP complex possesses an epitope that binds to T cells present in the individual, and thus whether the MAPP or higher order complex has potential use as a therapeutic composition for that individual. Suitable known assays for detection of the desired modulation (e.g., activation/proliferation or inhibition/suppression) of target T cells include, e.g., flow cytometric characterization of T cell phenotype, numbers, and/or antigen specificity. Such an assay to detect the presence of epitope-specific T cells, e.g., a companion diagnostic, can further include additional assays (e.g., effector cytokine ELISpot assays) and/or appropriate controls (e.g., antigen-specific and antigen-nonspecific multimeric peptide-HLA staining reagents) to determine whether the MAPP or higher order MAPP complex is selectively binding, modulating (activating or inhibiting), and/or expanding the target T cells. Thus, for example, 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, the method comprising: a) contacting in vitro the mixed population of T cells with a MAPP or higher order MAPP complex comprising an epitope of the present disclosure; and b) detecting modulation (activation or inhibition) and/or proliferation of T cells in response to said contacting, wherein modulation of and/or proliferation of T cells indicates the presence of the target T cell. Alternatively, and/or in addition, if activation and/or expansion (proliferation) of the desired T cell population is obtained using a MAPP or higher order MAPP complex (e.g., a duplex MAPP), then all or a portion of the population of T cells comprising the activated/expanded T cells can be administered back to the individual as a therapy.
In some instances, the population of T cells is in vivo in an individual. In such instances, a method of the present disclosure for selectively delivering a MOD polypeptide (e.g., PD-L1 or a reduced-affinity PD-L1) to an epitope-specific T cell comprises administering the MAPP or higher order MAPP complex (e.g., duplex MAPP) to the individual.
In some instances, the epitope-specific T cell to which a MOD polypeptide sequence (e.g., wild type or reduced affinity IL-2 and/or PD-L1 MOD) is being selectively delivered is referred to herein is a target regulatory T cell (Treg) that may inhibit or suppresses activity of an autoreactive T cell.
I. Dosages
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 MAPP (whether as a single heterodimer or, as described above, as a higher order complex such as a duplex MAPP) may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose; for example from 0.1 μg/kg body weight to 1.0 mg/kg body weight, from 0.1 mg/kg body weight to 0.5 mg/kg body weight, from 0.5 mg/kg body weight to 1 mg/kg body weight, from 1.0 mg/kg body weight to 5 mg/kg body weight, from 5 mg/kg body weight to 10 mg/kg body weight, from 10 mg/kg body weight to 15 mg/kg body weight, and from 15 mg/kg body weight to 20 mg/kg body weight. Doses below 0.1 mg/kg body weight or above 20 mg/kg are envisioned, especially considering the aforementioned factors. Amounts thus include from about 0.1 mg/kg body weight to about 0.5 mg/kg body weight, from about 0.5 mg/kg body weight to about 1 mg/kg body weight, from about 1.0 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, from about 15 mg/kg body weight to about 20 mg/kg body weight, and above about 20 mg/kg body weight.
Those of skill will readily appreciate that dose levels can vary as a function of the MAPP or higher order MAPP complex (e.g., duplex MAPP), 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.
In some cases, multiple doses of a MAPP or higher order MAPP complex (e.g., duplex MAPP) are administered. The frequency of administration of a MAPP or higher order MAPP complex (e.g., duplex MAPP) can vary depending on any of a variety of factors, e.g., severity of the symptoms, patient response, etc. For example, in some cases, a MAPP or higher order MAPP complex (e.g., duplex MAPP) is administered once per month, less frequently than once per month, e.g., once every 6 weeks, once every two months, once every three months, or more frequently than once per month, e.g., twice per month, three times per month, every other week (qow), one every three weeks, once every four weeks, once per week (qw), 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), daily (qd), twice a day (qid), or three times a day (tid).
The duration of administration of a MAPP, e.g., the period of time over which a MAPP is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, a MAPP can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about 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, including continued administration for the patient's life.
Where treatment is of a finite duration, following successful treatment, it may be desirable to have the patient undergo periodic maintenance therapy to prevent the recurrence of the disease state, wherein a MAPP is administered in maintenance doses, ranging from those recited above, i.e., 0.1 mg/kg body weight to about 0.5 mg/kg body weight, from about 0.5 mg/kg body weight to about 1 mg/kg body weight, from about 1.0 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, from about 15 mg/kg body weight to about 20 mg/kg body weight, and above about 20 mg/kg body weight. The periodic maintenance therapy can be once per month, once every two months, once every three months, once every four months, once every five months, once every six months, or less frequently than once every six months.
J. Routes of Administration
A MAPP or higher order MAPP complex (e.g., a duplex MAPP), or a nucleic acid or recombinant expression vectors comprising nucleic acids encoding one or more polypeptides of a MAPP or its higher order complexes, is administered to an individual using any available method and route suitable for drug delivery, including in vivo and in vitro methods, as well as systemic and localized routes of administration. A MAPP or higher order MAPP complex can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated for use in a method include, but are not necessarily limited to, enteral, parenteral, and inhalational routes.
Conventional and pharmaceutically acceptable routes of administration include intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intraarterial, intralymphatic, rectal, nasal, oral, intratumoral, peritumoral, and other enteral and parenteral routes of administration. Of these, intravenous, intramuscular and subcutaneous may be more commonly employed. MAPPS and their higher order complexes, nucleic acids and expression vectors encoding them may be administered, for example, intravenously. Routes of administration may be combined, if desired, or adjusted depending upon, for example, the MAPP or higher order MAPP complex (e.g., duplex MAPP) and/or the desired effect. A MAPP or higher order MAPP complex can be administered in a single dose or in multiple doses.
A MAPP or higher order MAPP complex (e.g., a duplex MAPP), or a nucleic acid or recombinant expression vectors comprising nucleic acids encoding one or more polypeptides of a MAPP or its higher order complexes may also be contacted with cells in vitro. The cells subject to such in vitro treatment and/or their progeny, may then be administered to a patient or subject (e.g., the subject from which the cells treated in vitro were obtained.
K. Subjects Suitable for Treatment
Subjects suitable for treatment include those with a cancer, infectious diseases (e.g., including those with viral, bacterial, and/or mycoplasma causative agents), allergic reactions, and/or autoimmune diseases.
Subjects suitable for treatment who have a cancer include, but are not limited to, individuals who have been provided other treatments for the cancer but who failed to respond to the treatment. Cancers that can be treated with a method include, but are not limited to, those displaying any of the cancer epitopes recited herein including, but not limited to peptide epitopes of AFP, WT-1, HPV and HBV.
Subjects suitable for treatment may also include individuals who have an infectious disease include, but are not limited to, individuals who have been provided other treatments for the infectious disease but who failed to respond to the treatment. Infectious diseases that can be treated with a method include, but are not limited to, those having an infectious agent recited herein including, but not limited to, EBV, HPV and HBV epitopes.
Subjects suitable for treatment also include individuals who have an allergy include, but are not limited to, individuals who have been provided other treatments for the allergy but who failed to respond to the treatment. Allergic conditions that can be treated with a method include, but are not limited to, those resulting from exposure to nuts (e.g., tree and/or peanuts), pollen, and insect venoms (e.g., bee and/or wasp venom antigens).
Subjects suitable for treatment include those with an autoimmune disease or a genetic disposition to develop an autoimmune disease including a family history of an autoimmune disease (e.g., a grandparent, parent, or sibling with the autoimmune disease. Subjects suitable for treatment who have an autoimmune disease include, but are not limited to, individuals who have been provided other treatments for the autoimmune disease but who failed to respond to the treatment. Autoimmune diseases that can be treated with a method include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune-associated infertility, autoimmune thrombocytopenic purpura, bullous pemphigoid, Celiac disease, Crohn's disease, Goodpasture's syndrome, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), Grave's disease, Hashimoto's thyroiditis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus (e.g., pemphigus vulgaris), pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus (SLE), Type 1 Diabetes, vasculitis, and vitiligo.
Certain aspects, including embodiments/aspects of the present subject matter described above, may be beneficial alone or in combination, with one or more other aspects recited hereinbelow. In addition, while the present disclosure has been disclosed with reference to certain aspects recited below and in the claims, numerous modifications, alterations, and changes to the described aspects/embodiments are possible without departing from the sphere and scope of the present disclosure. Accordingly, it is intended that the present disclosure not be limited to the described embodiments, aspects and claims, but that it has the full scope defined by the language of this disclosure and equivalents thereof.
YNQSX1A GSHTLQWMHG CELGPDX2RFL RGYEQFAYDG
A QISEQKSNDA SEAEHQX3X4YL
GDGT
X6
RG YNQSEA SSHTLQWMIX7 CDLX8X9DGRLX10 RGYEQYAYDG
A QISKRKCEAA NVAEQRRAX11L
GDGT
Example 1 illustrates three related MAPP heterodimer constructs that have multimerized into duplexes with the overall structures given in
The three duplex MAPPs in
Testing, conducted by contacting the MAPPs with a population of PBMCs obtained from one or more individuals that have not been exposed to the MART peptide epitope, demonstrates an expansion in the number of MART specific T cells as demonstrated by MART-tetramer staining In contrast, exposure of PBMC to MAPPs bearing an unrelated epitope does not increase MART specific T cell numbers.
Example 2 illustrates eight MAPP constructs that multimerized into duplexes due to interactions between antibody Fc multimerization sequences in the framework polypeptides, with the dimerization sequences pairing through CH1-Cκ light chain interactions. The constructs are made from six different framework polypeptides—4300, 4348, 4302, 4304, 4349, and 4550, that pair with one of six dimerization poly peptides—4345, 4346, 4294, 4296, 4347, and 4298. The sequences of those polypeptides are provided in
Construct 1 comprises two 4300 framework polypeptides and two 4345 dimerization polypeptides.
Construct 2 comprises two 4300 framework polypeptides and two 4346 dimerization polypeptides.
Construct 3 comprises two 4348 framework polypeptides and two 4294 dimerization polypeptides.
Construct 4 comprises two 4348 framework polypeptides and two 4296 dimerization polypeptides.
Construct 5 comprises two 4302 framework polypeptides and two 4347 dimerization polypeptides.
Construct 6 comprises two 4304 framework polypeptides and two 4347 dimerization polypeptides.
Construct 7 comprises two 4349 framework polypeptides and two 4298 dimerization polypeptides.
Construct 8 comprises two 4350 framework polypeptides and two 4298 dimerization polypeptides.
This application claims the benefit of U.S. Provisional Patent Appln. No. 63/120,671 filed Dec. 2, 2020, and the benefit of U.S. Provisional Patent Appln. No. 63/030,243 filed May 26, 2020. This application contains a sequence listing submitted electronically via EFS-web, which serves as both the paper copy and the computer readable form (CRF) and consists of a file entitled “123640-8015.WO00_seqlist.txt”, which was created on May 26, 2021, which is 502,390 bytes in size, and which is herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/034371 | 5/26/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63120671 | Dec 2020 | US | |
| 63030243 | May 2020 | US |
