Compositions and methods employing antibody mixtures

Abstract

The present disclosure relates to compositions and methods employing combinations of antibodies differing in their affinities for a target molecule and / or in their effector function to manage antibody-dependent cellular cytotoxicity (ADCC) and / or anti-body-dependent cellular phagocytosis (ADCP).

Description

Docket No. UM-44853.601COMPOSITIONS AND METHODS EMPLOYING ANTIBODY MIXTURESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Patent Application Serial Number 63 / 777, 137, filed March 25, 2025, the disclosure of which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The text of the computer readable sequence listing filed herewith, titled “44853-601_SEQUENCE_LISTING”, created March 24, 2026, having a file size of 9,238 bytes, is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERAL FUNDING

[0003] This invention was made with government support under W81XWH-21-1-0039 awarded by the Defense Health Agency, Medical Research and Development Branch. The government has certain rights in the invention.TECHNICAL FIELD

[0004] The present disclosure relates to compositions and methods employing combinations of antibodies differing in their affinities for a target molecule and / or in their effector function to manage antibody-dependent cellular cytotoxicity (ADCC) and / or anti-body-dependent cellular phagocytosis (ADCP).BACKGROUND

[0005] Cancer remains a major global health challenge, affecting millions and contributing to high mortality rates. Despite advances in early detection and treatment, it remains one of the leading causes of death. A key challenge in treatment is precisely targeting cancer cells while sparing healthy ones, as many therapies struggle with selectivity due to shared biological pathway s. This overlap makes it difficult to achieve effective "on-target" cancer cell destruction while minimizing "off-target" damage to healthy tissues, often resulting in systemic toxicity.

[0006] These side effects can reduce treatment effectiveness, requiring dose adjustments or discontinuation, particularly when adverse effects are severe. This compromises the therapeutic window, limiting the ability to deliver an effective dose while minimizing harm to healthy tissues.Docket No. UM-44853.601Furthermore, these toxicities can affect survivors' long-term health, raising the risk of secondary health issues years after treatment. Thus, there is an urgent need for a targeted treatment that minimizes toxicity on healthy cells while ensuring high efficacy of the therapeutic.SUMMARY

[0007] Provided herein are compositions and methods employing combinations of antibodies differing in their affinities for a target molecule and / or in their effector function to manage antibodydependent cellular cytotoxicity (ADCC) and / or anti-body-dependent cellular phagocytosis (ADCP). For example, provided herein are mixtures comprising a high avidity, low affinity (HALA) antibody and an effectorless or effector function-reduced, high-affinity antibody.

[0008] Fc-mediated effector functions are key for conferring potent antibody-mediated killing of cancer cells. However, it is difficult to achieve highly selective targeting of cancer cells while minimizing toxicity on healthy tissue because of tire expression of most receptors, albeit at lower levels, on non-cancer cells. Previous attempts to increase the selectivity of antibody-mediated effector functions have sought to reduce binding affinity and / or increase avidity, which typically results in modest improvements in selectivity. To overcome this limitation, the technology provided herein utilizes mixtures of antibodies that achieve high selectivity based on receptor level while maintaining high activity for cells with high receptor levels.

[0009] For example, experiments conducted during the development of embodiments of the technology utilized mixtures of two variants of an anti-HER2 antibody (trastuzumab), one that is affinity-reduced and effector-competent and a second high-affinity variant that is effectorless. It was observed that the high-affinity, effectorless antibody reduces effector function for cells with low receptor levels, including reduced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), while the high-avidity, effector-competent antibody mediates significant effector function for cells with high receptor levels. Moreover, replacing the effector-competent Fcregion of the affinity-reduced antibody with high-affinity Fcdomains that enhance effector function drives high activity while maintaining high selectivity for the antibody mixtures. These findings outline a general strategy for maximizing the therapeutic window by selectively targeting cells (e.g., cancer cells) based on receptor levels that find use for a wide range of applications involving antibody-mediated synapse formation, including antibody-drug conjugates and bispecific antibodies such as T cell engagers.

[0010] In some embodiments, provided herein are compositions (e.g., therapeutic compositions) comprising a first antibody and a second antibody that each bind to a cellular target molecule (e.g., a receptor, a receptor on a cancer cells, etc ), wherein the first antibody, compared to the second antibody, has a 10-fold or more (e.g., 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, 10,000-fold, etc.) lower affinity for the cellular target molecule. In someDocket No. UM-44853.601embodiments, the composition comprises a mixture of the first and second antibodies. In some embodiments, the first and / or second antibody can be any of a recombinant monoclonal antibody, an antibody fragment, a single-chain antibody, a bispecific or multi -specific antibody, or tire like.

[0011] Also provided herein are kits comprising such first and / or second antibodies. In some embodiments, the kit contains one or more containers each containing either one of or both the first and second antibodies.

[0012] In some embodiments, the second antibody has reduced effector function relative to the first antibody. In some embodiments, the second antibody has no effector function. In some embodiments, the second antibody has no Fcdomain. In some embodiments, the Fcdomain of the second antibody is a variant of the Fcdomain of the first antibody comprising one or more mutations that reduce or eliminate effector function. In some embodiments, the second antibody has reduced affinity for an Fcreceptor compared to said first antibody. In some embodiments, the Fcreceptor is one or more of FcyRI, FcyRII, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIII, FcyRIIIa or FcyRIIIb. In some embodiments, the Fcdomain comprises a sequence of SEQ ID NOs:8.

[0013] In some embodiments, the first antibody comprises a variant Fcregion that has enhanced effector function (e.g., relative to the second antibody or relative to an original starting antibody to which the first antibody is a variant). In some embodiments, the Fcregion comprises SEQ ID NOs: 6 or 7.

[0014] In some embodiments, the Fcdomain of the first or second antibody has one or more chemical modifications (e.g., glycosylation (e.g., reduced fucosylation, afucosylation); PEGylation: etc.) that alter effector function.

[0015] In some embodiments, the cellular target molecule comprises a cellular receptor. In some embodiments, the target molecule is overexpressed on cancer cells. In some embodiments, the cellular target molecule is human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), transferrin receptor (TfR), folate receptor (FR), an integrin receptor, a sialic acid receptor, a G-protein-coupled receptor (e.g., a chemokine receptor, a protease-activated receptor, a neuropeptide receptor, an adenosine A2B receptor, a P2Y purinoceptor, a calcium-sensing receptor, a metabotropic glutamate receptor), neurotensin receptor 1 (NTSR1), mesothelin (MSLN), prostate-specific membrane antigen (PSMA), and the like.

[0016] In some embodiments, the first and second antibodies are variants of antibody therapeutic agents that bind to targets overexpressed on cancer cells including, but not limited to. Trastuzumab (Herceptin), Pertuzumab (Pep eta). Margetuximab (Margenza), Ado-trastuzumab emtansine (T-DM1,Docket No. UM-44853.601Kadcyla), Cetuximab (Erbitux), Panitumumab (Vectibix), Rituximab (Rituxan), Obinutuzumab (Gazyva), Daratumumab (Darzalex), and Isatuximab (Sarclisa).

[0017] In some embodiments, the cellular target molecule is HER2 and the first antibody comprises SEQ ID Nos: 3 or 4.

[0018] Further provided herein are methods of treating a subject by administering the first and second antibody to the subject (e.g., administering one or more pharmaceutical compositions comprising the first and / or second antibodies). In some embodiments, the first antibody and second antibody are administered simultaneously to the subject. In some embodiments, the first antibody and second antibody are administered sequentially to the subject. In some embodiments, the second antibody is administered before the first antibody. In some embodiments, the first antibody is administered before the second antibody.

[0019] Also provided herein are uses of the first and second antibodies or compositions or kits comprising the first and second antibodies. In some embodiments, the use is to treat a disease or condition (e.g., cancer) having disease cells that overexpress the cellular target molecule. In some embodiments, the antibody-dependent cellular cytotoxicity (ADCC) selectivity and / or antibodydependent cellular phagocytosis (ADCP) selectivity are altered relative to a treatment with the first antibody alone or treatment with an antibody having the affinity of the second antibody and the effector function of the first antibody, or similar effector function.DEFINITIONS

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. Hie materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0021] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular fonns “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.Docket No. UM-44853.601

[0022] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1. 6.2. 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0023] The term “peptide” refers to an oligomer to short polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are of about 50 amino acids or less in length. A peptide may comprise natural amino acids, non-natural amino acids, amino acid analogs, and / or modified amino acids. A peptide may be a subsequence of naturally occurring protein or a non-natural sequence.

[0024] The terms “antibody” and “antibodies” as used herein refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi-specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C, et al., Nature Biotechnology 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology) 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH. or other domain structure), and functionally active epitope -binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte -binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4. IgAl, and IgA2), or subclass. For simplicity sake, an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody”.

[0025] The term “antibody fragment” as used herein refers to a portion of an intact antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-Docket No. UM-44853.6011129 (2005)) (e.g., comprises the antigen-binding site or variable region). Any antigen-binding fragment of the antibody described herein is within the scope of the present disclosure. The antibody may not include the constant heavy chain domains (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fcregion of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab')? fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0026] Typically, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the ‘‘hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2, and CHS) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct A es, either kappa (K) or lambda ( / .). based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0027] Tire variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N. Y. (2001)).

[0028] The term “CDR” is used herein to refer to the “complementarity determining region” within an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N-terminus of a heavy or light chain, these regions are denotedDocket No. UM-44853.601“CDR1,” “CDR2,” and “CDR3,” for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region that binds the antigen. An antigenbinding site, therefore, may include six CDRs. comprising the CDR set from each of a heavy and a light chain variable region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) may be referred to as a “molecular recognition unit.” Crystallographic analyses of antigen-antibody complexes have demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0029] The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as “Kabat CDRs”. Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); and Chothia et al., Nature, 342: 877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as “LI,” “L2,” and “L3,” or “Hl,” “H2,” and “H3,” where the “L” and the “H” designate the light chain and the heavy chain regions, respectively. These regions may be referred to as “Chothia CDRs,” which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan, FASEBJ., 9: 133-139 (1995), and MacCallum,. / . Mol. Biol., 262(5): 732-745 (1996). Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain aspects use Kabat- or Chothia-defined CDRs.

[0030] As used herein, when an antibody or other entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to facilitate detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (!<>,) of at least 107M1(e.g., >107M1,Docket No. UM-44853.601>108M1, >109M1, >1O10M1, >10nM1, >1012M1, >1013M1, etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.

[0031] The term “monoclonal antibody,’’ as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U. S. Patent 4,816,567). isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352: 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0032] The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

[0033] Tire term “bispecific” antibody as used herein denotes an antibody that has at least two binding sites each of which bind to different epitopes of the same antigen or a different antigen.

[0034] The term “multi specific” antibody as used herein denotes an antibody that has binding specificities for at least two different sites.

[0035] The term “valent” as used within the current application denotes the presence of a specified number of binding sites in an antibody molecule. As such, the terms “bivalent,” “tetravalent,” and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antibody molecule. The bispecific antibodies according to the invention are at least “bivalent” and may be “trivalent” or “multivalent” (e.g., “tetravalent” or “hexavalent”). That is, the antibodies may be bispecific even in cases where there are more than two binding sites (e.g., that the antibody is trivalent or multivalent).

[0036] The terms “immunogen” and “antigen” are used interchangeably herein and refer to any molecule, compound, or substance that induces an immune response in an animal (e.g., a mammal). An “immune response” can entail, for example, antibody production and / or the activation of immune effector cells. An antigen in the context of the disclosure can comprise any subunit, fragment, or epitope of any proteinaceous or non-proteinaceous (e.g., carbohydrate or lipid) molecule that provokesDocket No. UM-44853.601an immune response in a mammal. By “epitope” is meant a sequence of an antigen that is recognized by an antibody or an antigen receptor. Epitopes also are referred to in the art as “antigenic determinants.” In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. In certain embodiments, an epitope may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. Tn certain embodiments, an epitope may have specific three-dimensional structural characteristics (e.g., a “conformational” epitope) and / or specific charge characteristics. The antigen can be a protein or peptide of viral, bacterial, parasitic, fungal, protozoan, prion, cellular, or extracellular origin, which provokes an immune response in a mammal, preferably leading to protective immunity.

[0037] The terms “polynucleotide” or “oligonucleotide” or “nucleic acid,” as used herein, means at least two nucleotides covalently linked together. The polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxy ribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosinc and isoguanine. The nucleic acid, whether DNA or RNA may comprise non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”). Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. Polynucleotides may be single-or double-stranded or may contain portions of both double stranded and single stranded sequence. The depiction of a single strand also defines the sequence of the complementary strand. Tirus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.

[0038] The terms “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger. Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double -stranded fonn, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey,Docket No. UM-44853.601Biochemistry, 41(14): 4503-4510 (2002)) and U. S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt etal., Proc. Natl. Acad. Sci. U. S. A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc.. 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0039] The terms “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide” and “protein” are used interchangeably herein.

[0040] The term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by methods known in the art, including those described herein.

[0041] The term “avidity” refers to the overall stability or binding strength of the binding pair complex. The avidity depends on the affinity of a single binding site and its binding partner (e.g., epitope -antigen), and also all the other non-covalent interactions between the binding pair which are not directed to a single binding site (e.g., valency and structural arrangements). A common method for the estimation of avidity consists of the assessment of the stability of binding pair complexes in the presence of chaotropic agents. Avidity can also be measured by other methods known in the art, including those described herein which evaluate avidity using binding to an entire target versus the specific binding site. Avidity is dependent on the density of target (e.g., antigens) binding sites. For the carriers described herein, the avidity is high on cells or tissues with a high expression of the target but lower on cells or tissues with a low expression of the target.

[0042] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting to a target destination, such as, but not limited to, an organ, tissue, cell, or tumor, may occur by any means of administration known to the skilled artisan.Docket No. UM-44853.601

[0043] The terms ‘‘providing," “administering,” and “introducing” are used interchangeably herein and refer to placement into a subject by a method or route which results in at least partial localization to a desired site.

[0044] The terms “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0045] The terms “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a peptide or composition described herein to an appropriate subject. The term also includes a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the disease. As such, “treating” means an application or administration of the peptides or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.BRIEF DESCRIPTION OF THE FIGURES

[0046] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0047] FIG 1. shows binding affinity measurements for Fab fragments and IgGs corresponding to trastuzumab and its affinity-reduced mutants. (A) The panel of antibodies which represents wild-type trastuzumab (WT) and two affinity-reduced variants (S12 and S4) - was evaluated in the (B) Fab or (C-D) IgG formats for binding to (B-C) HER2+++(HCC1954) or (D) HER2 (MDA-MB-231) cells. The measurements were performed using flow cytometry, and the error bars are standard error (n=3).

[0048] FIG 2. shows analytical size-exclusion chromatography analysis of the Fab fragments and IgGs for wild-type trastuzumab and the HALA mutants. (A) WT Fab, (B) S12 Fab, (C) S4 Fab (D) WT IgG. (E) S12 IgG, and (F) S4 IgG.Docket No. UM-44853.601

[0049] FIG 3. shows ADCC reporter cell measurements for variants of trastuzumab with wildtype or FcγRIIIA affinity-enhanced Fcregions. (A-B) The fold activation of FcγRIIIA (ADCC reporter cells) for wild-type trastuzumab (WT), S12, and S4 with wild-type IgGl Fcregions corresponding to (A)HER2 and (B) HER2+cells, as well as (C) the ratio of HER2+++ / HER2+activities. (D-E) The fold activation for WT-eFcl, S12-eFcl, and S4-eFcl with a human IgG Fcregion containing mutations that enhance effector functions (eFcl) for (D) HER2+++and (E) HER2+cells, as well as (F) the ratio of HER2+++ / HER2+activities. (G-I) Comparison of the maximum activities relative to the maximum selectivities for (G) WT and WT-eFcl, (H) S12 and S12-eFcl, and (I) S4 and S4-eFcl. In (D-I), the mutated IgGl Fcregion (denoted as eFcl) contains five mutations in the CH2 and CH3 regions that enhance activity

[0038] , In (A-I), the fold activation is relative to signal without IgG, the data are averages of three independent experiments, and the error bars are standard errors.

[0050] FIG 4. shows statistical analysis of the selectivity for activating ADCC reporter cells for individual IgGs as a function of antibody concentration. (A-B) The ratio of HER2+++ / HER2+activities for antibodies with (A) wild-type or (B) enhanced Fc(eFcl) regions. Each experiment was performed three times (two replicates per experiment), the six data points are shown for each antibody concentration, and the errors are standard errors. The statistical significance was evaluated using a paired t-test, and the p- values are denoted as <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****).

[0051] FIG 5. shows proposed strategy to increase the selectivity of antibody-mediated effector function for target cells with elevated antigen expression using mixtures of engineered antibodies. (A) Wild-type trastuzumab, which has high affinity for its antigen (HER2), binds strongly to cells with either high (HER2+++) or low (HER2+) levels of antigen, and is contemplated to mediate relatively high effector function in both cases. This is expected to result in a relatively low effector function ratio for cells with high HER2 levels versus those with low HER2 levels. (B) An engineered version of trastuzumab, namely a High Avidity Low Affinity (HALA) variant, is contemplated to bind strongly to HER2+++cells and weakly to HER2+cells. This is expected to result in an increase in the effector function ratio, which is primarily due to reduced effector function on HER2+cells. (C) A mixture of the HALA antibody and an effectorless version of trastuzumab that has high HER2 affinity but little affinity for activating FcyRs - referred to as a blocking antibody - is expected to result in a high selectivity with moderate effector function ratios. (D) A mixture of the Fc-enhanced HALA antibody and the blocking antibody is expected to result in high effector function and selectivity. This is posited to be due to both the accumulation of the i) Fc-enhanced HALA antibody in the synapses involving HER2+++cells, which would lead to high effector function and ii) effectorless (high affinity) trastuzumab antibody in the synapses involving HER2+cells, which would lead to high selectivity.Docket No. UM-44853.601Together, the mixtures of the Fc-enhanced HALA antibody and effectorless, high-affinity blocking antibody are expected to lead to both high selectivity and effector function on HER2+++cells.

[0052] FIG 6. shows mixtures of HALA and blocker antibody variants of trastuzumab increase the specificity of HALA antibodies for HER2+++cells. (A-B) The binding signal of fluorescently-labeled wild-type trastuzumab (WT) or HALA variants (S12 and S4) with different ratios of an unlabeled blocking antibody (trastuzumab with Fcmutations that eliminate effector function) corresponding to (A) HER2+++and (B) HER2+cells, as well as (C) the ratio of binding for fluorescently-labeled WT trastuzumab at each antibody mixture concentration for HER2+++relative to HER2+cells. (D-E) Tire binding signal for fluorescently-labeled S12 with different ratios of the unlabeled blocking antibody corresponding to (D) HER2+++and (E) HER2+cells, as well as (F) the binding ratio. (G-H) The binding signal for fluorescently-labeled S4 with different ratios of the unlabeled blocking antibody corresponding to (G) HER2+++and (H) HER2+cells, as well as (I) the binding ratio.

[0053] FIG 7. shows statistical analysis of antibody binding selectivity as a function of HER2 expression level. (A-C) Binding selectivity (HER2+++ / HER2+cells) for fluorescently-labeled WT or HALA (S12 and S4) IgGs without or with blocker (i.e., unlabeled WT trastuzumab IgG) at equimolar levels for (A) WT, (B) S12, and (C) S4 IgGs. Each experiment was performed three times, the three data points are shown for each antibody concentration, and the errors are standard errors. The statistical significance was evaluated using a paired t-test, and the p-values are denoted as <0.05 (*), <0.01 (**), and <0.001 (***).

[0054] FIG 8. shows mixtures of HALA and blocker antibody variants of trastuzumab increase the selectivity for activating ADCC reporter cells based on the level of HER2 expression. (A-B) Hie fold activation of FcγRIIIA (ADCC reporter cells) for mixtures of wild-type trastuzumab (WT) and different ratios of a blocking antibody (trastuzumab with Fcmutations that eliminate effector function) corresponding to (A) HER2+++and (B) HER2+cells, as well as (C) the ratio of HER2+++ / HER2+activities. (D-E) The fold activation for mixtures of S12 with different ratios of the blocking antibody corresponding to (D) HER2+++and (E) HER2+cells, as well as (F) the ratio of activities. (G-H) The fold activation for mixtures of S4 with different ratios of the blocking antibody corresponding to (G) HER2+++and (H) HER2+cells, as well as (I) the ratio of activities. (J-L) Comparison of the maximum activities relative to the maximum selectivities for (J) WT: blocker mixtures, (K) S12:blocker mixtures, and (L) S4:blocker mixtures. In (A-L), the data are averages of three independent experiments, and the error bars are standard errors. In (K-L), the grey data and line correspond to the WT: blocker data shown in (J).Docket No. UM-44853.601

[0055] FIG 9. shows statistical analysis of antibody-mediated FcγRIIIA activation for wild-type trastuzumab and HALA variants alone and in the presence of a blocking antibody. (A-C) The fold activation of FcγRIIIA (ADCC reporter cells) for individual IgGs or equimolar mixtures thereof with a blocking antibody (trastuzumab with Fcmutations that eliminate effector function) corresponding to (A) HER2+++and (B) HER2+cells, as well as (C) the ratio of HER2+++ / HER2+activities. (D-F) The ratio of activities for individual IgGs or equimolar mixtures thereof involving a blocking antibody for (D) WT, (E) S12, and (F) S4 IgGs. Each experiment was performed three times (two replicates per experiment), the six data points are shown for each antibody concentration, and the errors are standard errors. The statistical significance was evaluated using a paired t-test, and the p-values are denoted as <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****).

[0056] FIG 10. shows mixtures of HALA antibodies with enhanced FcγRIIIA affinity and blocking antibodies maximize activity and selectivity while minimizing tradeoffs. (A-B) The fold activation of FcγRIIIA (ADCC reporter cells) for mixtures of wild-type trastuzumab with enhanced Fc1 (WT-eFc1) and different ratios of a blocking antibody corresponding to (A) HER2+++and (B) HER2+cells, as well as (C) the ratio of HER2+++ / HER2+activities. (D-E) The fold activation for mixtures of S12-eFcl with different ratios of the blocking antibody corresponding to (D) HER2+++and (E) HER2+cells, as well as (F) the ratio of activities. (G-H) The fold activation for mixtures of S4-eFc1 with different ratios of the blocking antibody corresponding to (G) HER2+++and (H) HER2+cells, as well as (I) the ratio of activities. (J-L) Comparison of the maximum activities relative to the maximum selectivities for (J) WT-eFc1:blocker mixtures, (K) S12-eFcl:blocker mixtures and (L) S4-eFc1:blocker mixtures. In (A-L), the data are averages of three independent experiments, and the error bars are standard errors. In (K-L), the grey data and line correspond to the WT-eFcl:blocker data shown in (J).

[0057] FIG 11. shows statistical analysis of antibody-mediated FcγRIIIA activation for trastuzumab variants with Fcmutations that enhance receptor activation, either alone or in combination with a blocking antibody. (A-C) The fold activation of FcyRIIIA (ADCC reporter cells) for individual IgGs with enhanced FcγRIIIA activity or equimolar mixtures thereof involving a blocking antibody corresponding to (A) HER2+++and (B) HER2+cells, as well as (C) the ratio of HER2 / HER2+activities. (D-F) The ratio of activities (HER2+++ / HER2+cells) for individual IgGs with enhanced FcγRIIIA activity or equimolar mixtures thereof involving a blocking antibody for (D) WT-eFcl, (E) S12-eFcl, and (F) S4-eFcl IgGs. Each experiment was perfonned three times (two replicates per experiment), the six data points are shown for each antibody concentration, and the errorsDocket No. UM-44853.601are standard errors. The statistical significance was evaluated using a paired t-test, and the p- values are denoted as <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****).

[0058] FIG 12. shows mixtures of HALA antibodies improve the selectivity of ADCC based on HER2 expression level. (A) The percentage of cancer cells lysed for S4-eFcl (10 nM) alone and with a 1: 1 blocker ratio for cell lines with different HER2 expression levels and effector cells with different FcγRIIIA allotypes. (B) The percentage reduction of cancer cell lysis corresponding to the results in (A). (C) The ratio of the percent lysis of cancer cells corresponding to those with high HER2 expression relative to low HER2+expression. In (A-C), the data are averages of two independent experiments, and the error bars are standard deviations.

[0059] FIG 13. shows ADCP reporter cell measurements for variants of trastuzumab with or without mutations that enhance affinity for FcyRIIA. (A-B) The fold activation of FcyRIIA (ADCP reporter cells) for wild-type trastuzumab (WT). S12 and S4 with wild-type IgGl Fcregions corresponding to (A) HER2+++and (B) HER2+cells, as well as (C) the ratio of HER2+++ / HER2+activities. (D-E) The fold activation of FcyRIIA for WT-eFc2, S12-eFc2 and S4-eFc2 with a human IgGl Fcregion containing three mutations that enhance effector functions (denoted as eFc2)

[0042] for (D) HER2+++and (E) HER2+cells, as well as (F) tire ratio of activities. (G-I) Comparison of the maximum activities relative to the maximum selectivities for (G) WT and WT-eFc2, (H) S12 and S12-eFc2 and (I) S4 and S4-eFc2. In (A-I), the fold activation of FcyRIIA is relative to signal without IgG, the data are averages of three independent experiments, and the error bars are standard errors.

[0060] FIG 14. shows ADCP reporter cell measurements for variants of trastuzumab with enhanced Fcl. (A-B) The fold activation of FcyRIIA for Fc-enhanced WT trastuzumab (WT-eFcl), S12-eFcl and S4-eFcl corresponding to (A) HER2+++and (B) HER2+cells, as well as (C) the ratio of HER2+++ / HER2+activities at each antibody concentration. The fold activation is relative to signal without IgG, the data are averages of three independent experiments, and the error bars are standard errors.

[0061] FIG 15. shows statistical analysis of the selectivity for activating FcyRIIA using individual IgGs as a function of antibody concentration. (A-B) The ratios of HER2+++ / HER2+activities for antibodies with (A) WT or (B) enhanced Fc(eFc2) regions. Each experiment was performed three times (two replicates per experiment), the six data points are shown for each antibody concentration, and the errors are standard errors. The statistical significance was evaluated using a paired t-test, and the p-values are denoted as <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****).

[0062] FIG 16. shows mixtures of HALA antibodies with enhanced FcyRIIA activity and blocking antibodies maximize activity and selectivity while minimizing tradeoffs. (A-B) The foldDocket No. UM-44853.601activation of FcyRIIA (ADCP reporter cells) for mixtures of wild-type trastuzumab with enhanced Fc2 (WT-eFc2) with different ratios of a blocking antibody corresponding to (A) HER2+++and (B) HER2+cells, as well as (C) the ratio of HER2+++ / HER2+activities. (D-E) The fold activation for mixtures of S12-eFc2 with different ratios of the blocking antibody corresponding to (D) HER2+++and (E) HER2 cells, as well as (F) the ratio of activities. (G-H) The fold activation for mixtures of S4-eFc2 with different ratios of the blocking antibody corresponding to (G) HER2+++and (H) HER2+cells, as well as (I) the ratio of activities. (J-L) Comparison of the maximum activities relative to the maximum selectivities for (J) WT-eFc2:blocker mixtures. (K) S12-eFc2:blocker mixtures and (L) S4-eFc2:blocker mixtures. In (A-L), the data are averages of three independent experiments, and the error bars are standard errors. In (K-L), the grey data and line correspond to the WT-eFc2:blocker data shown in (J).

[0063] FIG 17. shows statistical analysis of antibody-mediated FcyRIIA activation for trastuzumab variants with Fcmutations that enhance receptor activation, either alone or in combination with a blocking antibody. (A-C) The fold activation of FcγRIIA (ADCP reporter cells) for individual IgGs with enhanced FcγRIIA activity or equimolar mixtures thereof involving a blocking antibody for (A) HER2+++and (B) HER2+cells, as well as (C) the ADCP ratios. (D-F) The ratio of HER2+++ / HER2+activities for individual IgGs with enhanced FcyRIIA activity or equimolar mixtures thereof involving a blocking antibody for (D) WT-eFc2, (E) S12-eFc2, and (F) S4-eFc2 IgGs. Each experiment was performed three times (two replicates per experiment), the six data points are shown for each antibody concentration, and the errors are standard errors. The statistical significance was evaluated using a paired t-test, and the p-values are denoted as <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****).

[0064] FIG 18. shows mixtures of HALA antibodies improve the selectivity of ADCP based on HER2 expression level. (A) The percentage of cancer cell phagocytosed for S4-eFc2 (10 nM) alone and with a 1:1 blocker ratio for cell lines with different HER2 expression levels and effector cells with different FcyRIIA allotypes. (B) The percentage reduction of cancer cell phagocytosis corresponding to the results in (A). In (A-B), the data are averages of two independent experiments, and the error bars are standard deviations.DETAILED DESCRIPTION

[0065] Monoclonal antibodies (mAbs) are widely used as cancer therapeutics for targeting both cancer cells and immune receptors. A critical part of their therapeutic activity is due to their Fc-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibodydependent cellular phagocytosis (ADCP) [1], For example, ADCC has been shown to play anDocket No. UM-44853.601important role in the cytotoxic activity of several antibody therapeutics, including antibodies against tumor-associated antigens such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and B-lymphocyte antigen CD20 [2-5]. ADCC also contributes to antibodies targeting immune checkpoints - such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) - by clearing regulatory T cells [6], ADCP has also been shown to play an important role in therapeutic efficacy for multiple antibodies, including those against CD20 and CD38 [7, 8],

[0066] Given the importance of such effector activity, there have been significant efforts to enhance antibody-mediated effector functions. One approach is to engineer the Fcregion to more strongly bind to activating Fcy receptors and more weakly to inhibitory receptors, which has been used to enhance the effector functions of the anti-HER2 antibody margetuximab [9, 10], A second approach is to improve Fcbinding to Fcy receptors via afucosylation, as employed by the antibody-drug conjugate (ADC) belantamab mafodotin

[0011] . A third approach is not to rely on the Fcregion for mediating the primary effector function, but rather use bispecific antibodies to recruit T cells to kill cancer cells. For example, bispecific antibodies that link CD3 engagement on T cells with various cancer-associated antigens, including CD 19, Carcinoembryonic antigen (CEA), and HER2, have shown promising and potent anti -cancer activities [12-14],

[0067] Despite the great promise of using antibodies to elicit potent effector functions, the problem of on-target, off-tumor toxicity continues to be a major challenge. For example, some mAb treatments lead to strong side effects due to this type of toxicity, including cutaneous toxicity (cetuximab), cardiotoxicity (trastuzumab), proteinuria and hypertension (bevacizumab), and neuropathic pain (dinutuximab and naxitamab) [15-19], This same type of problem also occurs for mAbs used in other therapeutic applications, such as ADCs and T cell-engaging bispecific antibodies. For example, ADCs directed against CD44v6 (bivatuzumab mertansine) and Nectin-4 (Padcev) cause severe skin toxicity [20, 21], Moreover, a bispecific T cell engager targeting CD3 and EGFR to treat colorectal cancer cells resulted in cytokine release and damage to EGFR-expressing organs by redirected T cells

[0022] ,

[0068] Many creative approaches have been reported for overcoming on-target, off-tumor toxicity issues. For example, reduction of antibody affinity has been successfully employed to reduce off-tumor binding for CAR-T cells while maintaining on-tumor cytotoxic activity

[0023] , CAR-T cells targeting CD38 with -1000-fold reduced affinity were able to lyse multiple myeloma cells (high CD38 expression) with similar efficiency as its high-affinity counterpart, but only the low-affinity variant spared healthy hematopoietic cells (low CD38 expression). Similarly, a moderate affinity EpCAM targeting antibody showed a larger therapeutic window compared to other higher affinity antibodies that caused acute pancreatitis

[0024] , A -40-fold reduction in affinity of an anti-MET ADC resulted in reduced liver toxicity while maintaining similar tumor uptake and efficacy

[0025] , If a specific Fc-Docket No. UM-44853.601effector function contributes to toxicity but not efficacy, Fcmutations can tailor the effector function to improve the therapeutic window

[0026] . This approach of reducing affinity has also been coupled with increasing antibody valency to further expand the therapeutic window for bispecific antibodies. For example, avidity-based binding to HER2 using bispecific antibodies with two binding sites for HER2 and one binding site for CD3 resulted in more selective killing of HER2 -overexpressing cells for variants with low HER2 affinity relative to those with high HER2 affinity

[0014] ,

[0069] A related study aimed at improving the in vivo selectivity of bispecific antibody targeting of model cancer cells with two targets (HER2 and EGFR) relative to cells with only one target (HER2) found that reducing both EGFR antibody avidity (i.e., monovalent Fab binding) and affinity for a 1x1 HER2 / EGFR bispecific antibody (i.e., one Fab specific for HER2 and one Fab specific for EGFR) was necessary to significantly increase targeting selectivity

[0027] , Moreover, it has also been shown that reducing the affinity of antibody Fcregions for FcyRs can reduce toxicity, such as liver toxicity associated with CD137 antibodies

[0028] , Finally, a different approach to reduce target engagement on healthy cells is to engineer the Fv or Fcregions of antibodies to preferentially bind to their corresponding targets (i.e., antigen or Fcreceptor, respectively) at the reduced pH conditions in the tumor microenvironment, such as pH-dependent binding for i) the Fab region binding to HER2 or EGFR [29, 30] or ii) the Fcregion binding to FcyRIII

[0031] ,

[0070] However, these previous approaches suffer from two key inherent limitations. First, attempts to increase the selectivity of antibody targeting of tumor vs. non-tumor cells typically result in reduced maximal cell-killing activity. Second, approaches that use bispecific antibodies - such as T-cell engagers - typically use non-standard antibody formats, which suffer from increased antibody complexity and associated risks related to increased developability and manufacturing challenges.

[0071] The technology provided herein addresses both previous limitations, which is summarized in Fig. 1 and Fig. 5. This approach can be used to improve the performance of existing antibodies by generating mixtures comprising two different variants of the antibody. Alternatively, new antibodies may be generated and used in mixtures to achieve the desired result. In some embodiments, this approach uses an antibody, which is sometimes referred to herein as a “blocking antibody,” that maintains high affinity for the target but contains mutations in the Fcregion, or a deletion of the Fcregion, to reduce or eliminate effector function, thereby blocking antibody activity on low-expressing cells. The different antibody, which has intact or enhanced Fceffector functions, has affinity-reducing mutations (e.g.. in the Fab regions) to reduce antibody binding to cells with low receptor levels while maintaining binding to cells with high receptor levels. Herein, it is demonstrated that the combinations of the two antibodies permits both high selectivity and effector function activity (ADCC and ADCP) for cells with high receptor levels relative to those with low receptor levels.Docket No. UM-44853.601

[0072] The binding affinity of therapeutic antibodies plays a role in determining their efficacy, specificity, and overall therapeutic potential. By selecting the affinities of first and second antibodies to a cellular target molecule, the present invention provides enhanced selectivity, binding avidity and functional activity for improved treatment outcomes. In some embodiments, the first antibody has a lower binding affinity, compared to the second antibody, to the cellular target molecule while maintaining target specificity. For example, the first antibody may have affinity-reducing mutations in the Fab regions to reduce antibody binding to cells with low levels of cellular target molecules while maintaining binding to cells with high levels of cellular target molecules.

[0073] Affinity, in the context of receptor binding, can be assessed by the strength of the interaction between a single heavy chain (HC) and / or light chain (LC) of an antibody and its target receptor. It is quantitatively represented by the dissociation constant (KD), which reflects the equilibrium between bound and unbound states of the antibody and receptor.

[0074] In some embodiments, the second antibody has a higher binding affinity, compared to the first antibody, to the cellular target molecule. In some embodiments, the binding affinity for the first antibody, compared to the second antibody is lower, e g., by at least 2-fold, at least 5 -fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10,000-fold, etc., against the cellular target molecule. The affinity of the second antibody may be selected based on the internalization rate of the target rather than strictly based on the affinity of the first antibody. As such, the relative affinity of the second antibody to the first antibody may be selected to be high if the affinity of the first antibody is high (e.g., a 300 nM Kd of the 2ndantibody might be selected whether the first antibody is 1 nM Kd or 30 pM Kd, in the latter case having an affinity ratio of 10,000-fold versus 300-fold in the former). Further, other factors may be considered in selecting relative or absolute affinities, including crosslinking binding, which may bring about additional avidity from a second cell in the synapse. For example, a particular affinity ratio based on a binding assay may be higher than an associated measured ADCC due to an avidity boost from high affinity Fc-crosslinking between a cancer cell and an effector cell.

[0075] Affinity can be measured by methods known in the art, including those described herein. Binding affinity can be assessed by measuring the interaction between an isolated heavy or light chain and its respective target antigen or receptor. This is often achieved using biophysical techniques such as Surface Plasmon Resonance (SPR). Isothermal Titration Calorimetry (ITC), fluorescence assays, radiolabeled assays, or Bio-Layer Interferometry (BLI), which provide real-time kinetic measurements of association and dissociation rates. Additionally, enzyme-linked immunosorbent assays (ELISA) can be employed to compare relative affinities by determining the concentration at which half-maximal binding occurs.Docket No. UM-44853.601

[0076] To compare the binding affinities of different antibody variants, standardized competition assays, kinetic analysis, and equilibrium binding studies are conducted. SPR allows direct comparison by analyzing changes in association and dissociation rates for each variant, providing a ranking of binding strengths. Similarly, flow cytometry-based receptor binding assays can quantify relative affinities in a cellular context, distinguishing between high-, moderate-, and low-affinity interactions based on fluorescence intensity shifts. Comparative affinity data can be used to optimize antibody engineering by selecting variants with desirable binding properties for therapeutic or diagnostic applications.

[0077] Effector function modulation refers to the alteration of an antibody’s ability to engage immune system components through modifications in its Fc(fragment crystallizable) region. By introducing specific amino acid substitutions or glycoengineering approaches, effector function can be enhanced, reduced, or completely eliminated, allowing for greater control over immune activation in therapeutic applications. These modifications permit antibodies to be optimized for specific clinical applications, such as enhancing anti-tumor activity, reducing off-target immune activation, or modulating immune responses.

[0078] In some embodiments, an Fcregion of an antibody is modified through specific amino acid mutations in the Fcregion by enhancing or reducing binding to an Fcreceptor (c.g., FcyR). In some embodiments, an Fcregion of an antibody is modified through glycoengineering. For example, altering glycosylation sites in the Fcdomain to increase or reduce complement activation. In some embodiments, an Fcregion of an antibody is modified through Fcreceptor affinity engineering; for example, modifying the Fcreceptor-binding sites (such as FcyRI, FcyRII, or FcγRIII) to either enhance or diminish binding affinity for immune cells activation. In some embodiments, an Fcregion of an antibody is modified through modifying the hinge region of the antibody. In some embodiments, an Fcregion of an antibody is modified through removal of the Fcdomain.

[0079] In some embodiments, the first and second antibodies may comprise an engineered Fcdomain. In some embodiments, the first antibody may comprise an engineered Fcdomain comprising one or more variant amino acid sequences or modifications that enhances effector function. In some embodiments, the second antibody may comprise an engineered Fcdomain comprising one or more variant amino acid sequences or modifications that reduces or eliminates effector function. In some embodiments, the second antibody has an engineered Fcdomain that renders it effectorless. In some embodiments, the second antibody has a reduced effector function relative to the first antibody. In some embodiments, the second antibody has no effector function. In some embodiments, the second antibody has no Fcdomain.Docket No. UM-44853.601

[0080] In some embodiments, the first and second antibodies comprise engineered Fcdomain for differential binding affinities to target cells expressing varying levels of cellular target molecules. In some embodiments, these variants include one or more amino acid modifications in the CH2 and CH3 regions of the Fcdomain to enhance or modulate antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) functions. For example, the first and second antibodies may comprise engineered Fcdomains to modulate antibody-dependent cell-mediated cytotoxicity (ADCC), antibodydependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cytokine release (ADCR). In certain embodiments, the Fcdomain modifications include, but are not limited to, amino acid substitutions that improve Fcy receptor binding, thereby enhancing immune effector activity. In certain embodiments, the Fcdomain modifications include, but are not limited to, amino acid substitutions that reduce or eliminate Fcy receptor binding, thereby reducing or eliminating immune effector activity. In some embodiments, the first and second antibodies may comprise an engineered Fcdomains to selectively engage with specific Fcreceptors (e.g., to bind to FcyRIIa and FcyRIIIa but not to FcyRI). In some embodiments, the first antibody’s Fcdomain binds to one or more Fcreceptors. In some embodiments, the second antibody’s Fcdomain binds to no Fcreceptors. In some embodiments, the Fcreceptors include but, are not limited to, FcyRI, FcyRII, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIII, FcyRIIIa or FcyRIIIb.

[0081] The therapeutic compositions are not limited by the quantity or concentration of the first antibody or the second antibody. In some embodiments, the compositions comprise an equimolar or molar excess of the first antibody to the second antibody or the second antibody to the first antibody. Thus, the compositions may comprise at least 1.1 fold (e.g., 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, or more) higher concentration of the first or second antibody to the other. In some embodiments, the compositions have a molar ratio of the first antibody to the second antibody of 1:1, less than 1:1 (e.g., at least 1:2, at least 1:3, at least 1:4, at least 1:5, at least 1:10, or more) or more than 1:1 (e.g., at least 2:1. at least 3:1. at least 4:1, at least 5:1, at least 10:1, or more).

[0082] The therapeutic compositions may comprise excipients or pharmaceutically acceptable carriers. Tire choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. The compositions of the present invention will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

[0083] The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, surfactant, cyclodextrins or formulationDocket No. UM-44853.601auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, com starch and potato starch: cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth: malt: gelatin; talc: excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; surfactants such as, but not limited to, cremophor EL, cremophor RH 60, Solutol HS 15 and polysorbate 80: cyclodextrins such as, but not limited to, alpha-CD, beta-CD, gamma-CD, HP-beta-CD, SBE-beta-CD: glycols; such as propylene glycol; esters such as, but not limited to. ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as releasing agents, coating agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0084] The route by which the disclosed compounds are administered, and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral (e.g., intradermal (IM), subcutaneous (SQ), intramuscular (IM), and intravenous (IV)) injections) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).

[0085] Any of the above compositions or formulations disclosed herein may further comprise at least one additional therapeutic agent. In some embodiments the at least one additional therapeutic agent comprises an immune modulator, a chemotherapeutic agent, a nucleic acid (e.g., mRNA, aptamers, antisense oligonucleotides, ribozyme nucleic acids, interfering RNAs, antisense and antigene nucleic acids), a steroid, an analgesic, an immunotherapy, or a combination thereof.

[0086] In some embodiments, the at least one additional therapeutic agent comprises at least one chemotherapeutic agent. As used herein, the term “chemotherapeutic” or “anti-cancer drug” includes any small molecule or other drug used in cancer treatment or prevention. Chemotherapeutics include, but are not limited to, cyclophosphamide, methotrexate, 5 -fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, afinitor (Everolimus), alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene,Docket No. UM-44853.601fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, and tucatinib.

[0087] In some embodiments, the at least one additional therapeutic agent comprises a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term “polynucleotide,” in its broadest sense, includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary' polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc.

[0088] In some embodiments, the at least one additional therapeutic agent is an RNA. RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to. shortmers, antagomirs, antisense RNAs, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA). microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof.

[0089] Dysregulation of cellular receptors and their associated signaling pathways, through one of the mechanisms, can lead to various human disorders. These include cancer, cardiovascular diseases, neurological disorders, metabolic and endocrine disorders, autoimmune diseases, and infectious diseases. Antibody therapies that target overexpressed cellular targets have been developed and are in development for such diseases and conditions. The antibody mixture and design strategy described herein finds use as an enhanced therapeutic option for addressing these diseases and conditions.

[0090] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a blood cancer or lymphoma. In some embodiments, the cancer is metastatic cancer. In some embodiments, the disclosed compounds, compositions, or methods result in suppression of elimination of metastasis. In some embodiments, the disclosed compounds, compositions, or methods result in decreased tumor growth. In some embodiments, the disclosed compounds, compositions, or methods prevent tumor recurrence.

[0091] Tire compounds and compositions herein may be useful to treat a wide variety of cancers including carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon / rectum, endometrium, head and neck, kidney, liver, lung, lymph nodes, muscle tissue, ovary, pancreas,Docket No. UM-44853.601prostate, skin, spleen, stomach, testicle, thyroid, or uterus. In select embodiments, the cancer is breast cancer.

[0092] In another aspect, the disclosure provides kits comprising at least one first antibody and at least one second antibody, as described above, or a composition comprising the at least at least one first antibody and at least one second antibody, and instructions for use.

[0093] The kits can also comprise other agents and / or products co-packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising another agent for delivery to a patient.

[0094] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background infomiation, list of components, brief or detailed protocols for using the compositions, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0095] It is understood that the disclosed kits can be employed in connection with the disclosed methods. Hie kit may further contain containers or devices for use with the methods or compositions disclosed herein. The kits optionally may provide additional components such as buffers and disposable single-use equipment. For example, the kits can comprise delivery- devices (e.g., syringes).

[0096] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.Example 1:Materials and Methods

[0097] Cloning and recombinant antibody expression. For trastuzumab mutants with wild-type Fc, VH genes were digested with EcoRI-HF (NEB, R3101L), and Nhel-HF (NEB, R3131L), VL genes were digested with EcoRI-HF and BsiWI-HF (NEB, R3553L). pTT5 mammalian expression plasmids containing a common human IgGl heavy or light chain (kappa) framework were digested with appropriate restriction enzymes and treated with calf intestinal alkaline phosphatase (CIP) (NEB, M0525L). Digested inserts and vectors were analyzed by electrophoresis using a 1% agarose gel, purified by Qiagen Gel Extraction protocol, ligated with T4 ligase (NEB, M0202L), and transformed by heat shock into DH5a E. coli cells. Sequences were confirmed by Sanger sequencing. Fab fragmentsDocket No. UM-44853.601of trastuzumab mutants were similarly cloned into modified pTT5 mammalian expression plasmids containing IgGl CHI or CL1 framework (linked with a His-tag).

[0098] For trastuzumab mutants with mutated Fc, enhancing Fcfragments were ordered as gBlocks (IDT) and replaced the wild-type Fcby overlap PCR. Five mutations (L235V, F243L, R292P, Y300L, and P396L) were introduced for eFcl, three mutations (G236A, S239D, I332E) were introduced as eFc2, and three mutations (L234A, L235A, and P329G) were introduced as effectorless Fc. The new heavy chains were digested with EcoRI-HF (NEB, R3101L) and BamHI-HF (R3136L) and inserted into pTT5 mammalian expression plasmids.

[0099] Shown below are the sequences for the VH and VL domains of trastuzumab and the VL domain of two mutants (S12 and S4). The VL domain for two mutants (S12 and S4) is the same as that of trastuzumab. The CDRs are shown in italics for each domain. Additionally, shown below are the sequences for the mutated Fcfragments — eFcl (five mutations), eFc2 (three mutations), and an effectorless Fc(three mutations).

[0100] Trastuzumab VH:EV QLVESGGGLV QPGGSLRLSC AASGFNIKDTYIHWVRQAPGKGLEWVAJUYPTNGYTRYAD SU TGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRlFGGDGFE DyWGQGTLVTVSS (SEQ ID NO: 1)

[0101] Trastuzumab VL:

[0102] DIQMTQSPSSI. SAS VGDR VTITC / d. S'O / ) IA7d VA WYQQK PGK APKI. EIY. S4. S7-7. ESG VP SRFSGSRSGTDFTLTISSLQPEDFATYYCOgTOTTRPTFGQGTKVEIK (SEQ ID NO: 2)

[0103] S4 VH:EVQLVESGGGLVQPGGSLRLSCAASG7'A7 '7)7'F / / 7WVRQAPGKGLE\VVA7 / F / '. SA'GF77? E47). S' 1AGRFTIS ADTS KNTA YLQMNS LRAEDTA VY YC. S7 WYGVAA FEMDTWGQGTLVTVSS (SEQ ID NO: 3)

[0104] S12 VH:

[0105] EVQLVESGGGLVQPGGSLRLSCAASG7'A7k'7)777 / / WVRQAPGKGLEWVA7 / I77VA'GF r FAOSFXGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRIFDAAGFFWDyWGQGTLVTV SS (SEQ ID NO: 4)

[0106] Reference IgG Fc:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:5)Docket No. UM-44853.601

[0107] eFcl mutation 1: L235V

[0108] eFcl mutation 2: F243L

[0109] eFcl mutation 3: R292P

[0110] eFcl mutation 4: Y300L

[0111] eFcl mutation 5: P396L

[0112] eFd:APELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP PEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:6)

[0113] eFc2 mutation 1: G236A

[0114] eFc2 mutation 2: S239D

[0115] eFc2 mutation 3: I332E

[0116] eFc2:APELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:7)

[0117] effectorless Fcmutation 1: L234A

[0118] effectorless Fcmutation 2: L235A

[0119] effectorless Fcmutation 3: P329G

[0120] effectorless:APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8)

[0121] For IgG and Fab expression, the CHO-3E7 cell line (L- 11992, National Research Council Canada) was cultured in 50 ml disposable conical tubes with F17 (50591354, Thermo Fisher Scientific) media with 4 mM L-glutamine and 0.1% Kolliphor Pl 88. The cultures were incubated at 37 °C and 250 rpm. Soluble IgGs were produced via transient transfection (30 ml) using 12.5 pg each of heavy and light chain plasmids mixed with 100 pg Polyethylenimine (PEI MAX, 247651, Polysciences Inc.). After transfection, 15 mM Glucose, 1.0% Tryptone N1 (TNI) (TekniScience,Docket No. UM-44853.60119553) and 0.5 mM Valproic Acid (VP A) (Sigma, P4543) were added to each batch. Six days after transfection, cultures were centrifuged at 3500 x g for 40 min. Supernatants were collected into new tubes.

[0122] For IgG purification, 0.5 ml dry volume of Protein A beads (Thermo Fisher Scientific, 20333) per tube was added. Mixtures were gently shaken at 4 °C overnight. Protein A beads were then collected by vacuum filter columns (Thermo Fisher Scientific, 89898) and then washed with around 100 ml of PBS. Antibodies were then eluted by 1 ml 0.1 M glycine buffer (pH 3.0) and then was buffer exchanged to 0.1 M acetate (PH 5.0) with Zeba desalting columns (Thermo Fisher Scientific. 89890). For Fab fragments, 0.5 ml dry volume ofNi-NTA beads (Qiagen, 30210) per tube was added. Mixtures were gently shaken at 4 °C overnight. Beads were collected by vacuum filter columns and then washed with around 100 ml of PBS. Fab fragments were washed with 50 mM imidazole and eluted with 1 ml 100 mM imidazole, then buffer-exchanged to 0.1 M acetate (pH 5.0). After measuring the concentration, IgGs and Fab fragments were aliquoted and stored at -80 °C.

[0123] IgG and Fab fragments were further purified by preparative size-exclusion chromatography (SEC) using a Shimadzu Prominence semi-prep HPLC System outfitted with an LC-20AT pump, SIL-20AC autosampler, and FRC-10A fraction collector. Proteins were loaded onto an SEC column (Superdex 200 Increase 10 / 300 GL column; GE, 28990944) and analyzed at 0.75 ml / minute using a PBS running buffer with 200 mM arginine (pH 7.4). After purification, IgG and Fab fragments were buffer exchanged into PBS with Zeba desalting columns (89890, Thermo Fisher), aliquoted, snap frozen, and stored at -80 °C.

[0124] Antibody fluorescent labeling. Fab fragments or IgG were concentrated to -2 mg / ml in 100 ml PBS in 1.5 ml tubes. 10 pL of 7.5% sodium bicarbonate (Millipore Sigma S6014) was add to each tube to make sure pH is around 8-8.5. Then 2 ul of 10 mg / ml Alexa-647 (Fisher Scientific A37573) was add to each tube. Tubes were put on a shaking plate for 1 hour at room temperature. To purify the fluorescently labeled Fab fragments. 850 pL of 10% P6 Biogel (Bio-Rad 1504130) was added to a costar spin-x tube (Coming 07-200-387) and spun down at 3500 x g for 1.5 minutes. The eluent from the bottom of the tube was removed and then -400 pL more 10% P6 Biogel was added again. Tubes were spun down at 3500 x g for 1.5 minutes. Tire eluent from the bottom of the tube was removed and the reaction mixture was added to the top of the column. Tubes were spun down at 3500 x g for 1.5 minutes. The eluent was collected into a new 1.5 ml tube. The protein concentration and degree of labeling were measured on the nanodrop.

[0125] Fab affinity measurements. HCC1954 cells (ATCC, CRL-2338) were cultured in T75 flasks using RPMI 1640 media (Fisher A 1049101) with 10% HIFBS (Fisher 10082147) and 1% Penicillin-Streptomycin (P / S) (Fisher 15140122) at 37 °C and 5% COz. HCC1954 cells in each T75 were treatedDocket No. UM-44853.601with 2.5 ml trypsin (Fisher 25-300-054). After detaching, 7.5 ml of RPMI 1640 was added and all media were collected in 15 ml tubes. Cells were spun down at 800 x g for 5 minutes and then rewashed and resuspended by Phosphate Buffered Saline with 0.1% Bovine Serum Albumin (PBSB). Fab fragments were diluted in PBSB as a 3-fold series starting from 1200 nM and added to 96 well u-bottom plates as 100 ul / well. Cells were added as 50,000 cellsAvell in 100 pL. Plates were then chilled on ice for 4 hours. After incubation, cells were washed with PBSB at 2500 x g for 5 minutes and then resuspended in 100 pL PBSB. Fluorescent signals were measured by flow cytometry (Bio-Rad Ze5).

[0126] IgG affinity measurement. HCC1954 cells were cultured in T75 flasks with RPMI 1640 media 10% HIFBS and 1% P / S at 37 °C and 5% CO2. MDA-MB-231 cells were cultured in T75 flasks using DMEM media (Fisher 11965092) with 10% HIFBS and 1% P / S at 37 °C and 5% CO2. IgGs were diluted in PBSB as a 3-fold series starting from 200 nM and added to 96 well u-bottom plates as 100 ul / well. Cells were added as 50,000 cells / well in 100 pL. Plates were then chilled on ice for 4 hours. After incubation, cells were washed with PBSB at 2500 x g for 5 minutes. Alexa anti-human Fc(Jackson Immuno Research 109-605-190) was diluted with PBSB 300-fold and added to each well as 100 pL. Plates were incubated on ice for 4 minutes. Then cells were washed with PBSB at 2500 x g for 5 minutes and then resuspended in 100 pL PBSB. Fluorescent signals were measured by flow cytometry.

[0127] IgG competitive binding measurement. HCC1954 cells and MDA-MB-231 cells were cultured. After detaching, cells were seeded to 96 well Tc-treated flat-bottom plates as 50,000 cells / well and incubated at 37 °C and 5% CO2 overnight. On the Second day, fluorescent-labeled antibodies were diluted as a 3-fold series starting from 100 nM, and blocking antibodies were added as the required ratio if needed. 100 pL of diluted antibody was added to each well and incubated at 37 °C and 5% CO2for 6 hours. After incubation, cells were detached by try psin and washed with PBS. Fluorescent signals were then measured by flow cytometry.

[0128] Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) reporter cell assays. ADCC cells (Invivogen jktl-nfat-cdl6) and ADCP cells (Invivogen jktl-nfat-cd32) were cultured in IMDM media (Fisher 12440053) with 10% HI FBS, 1% P / S, and 100 pg / ml Normocin (Invivogen ant-nr-1). 10 pg / ml of Blasticidin (Invivogen ant-bl-05) and 100 pg / ml of Zeocin (Invivogen ant-zn-05) were added to the growth media every’ passage. HCC-1954 and MDA-MB-231 cells were seeded to 96 well Tc-treated flat-bottom plates as 50,000 cells / well and incubated at 37 °C and 5% CO2 overnight. On the second day, testing antibodies were diluted as a 3 -fold series starting from 100 nM, and blocking antibodies were added as the required ratio if needed. Next, 110 pL of the diluted antibody? was added to each well and incubated at 37 °C and 5% CO2for 1 hour. The ADCC or ADCP cells were then centrifuged at 300 x g for 5Docket No. UM-44853.601minutes. The supernatant was removed and the ADCC cells were resuspended in the test medium (IMDM media with 10% HI FBS, 1% P / S). Ninety microliters of ADCC or ADCP cells (0.2 million cells) per well were added and the plates were incubated at 37 °C and 5% CO2 for 6 hours. After incubation, 20 pL of co-incubated supernatant was transferred into a white assay plate (Fisher 720091). Finally, 50 pL of QUANTI-Luc™ (Invivogen REPQLC2) was added per well and evaluated immediately with the luminometer measurement. Raw signals were normalized by the signal of negative controls (mixture of cancer cells and reporter cells without adding antibodies).

[0129] ADCC assay. PBMCs were purchased from STEMCELL (70025) and cultured in T-75 flasks with RPMI1640 (10% HI FBS). PBMC and target cells were then mixed with an effector (E): tumor (T) ratio of 10: 1, along with the indicated concentrations of antibodies at 37 °C and 5% CO2. After 4 h, the lactate dehydrogenase (LDH) releasing level was measured by CyQUANT Cytotoxicity Assay Kit (Fisher, C20301). The % lysis was calculated as (A-B) / (C-B) *100, where (A) is the background subtracted absorbance value of each test sample, (B) is the background subtracted absorbance value without effector cells (i.e., spontaneous release), and (C) is the background subtracted absorbance value for maximum cell lysis (i.e., adding lysis buffer to cancer cells without effector cells).

[0130] ADCP assay. PBMCs were purchased from STEMCELL (70025) and were cultured in T-75 flasks with RPMI1640 (10% HI FBS and 1% P / S) for 1 hour to allow attachment of the monocytes. Monocytes were then differentiated over 6 d in RPMI1640 with 100 ng / mL human M-CSF (PHC9501, Fisher). 10% HI FBS. and 1% P / S. Macrophages were labeled with CellTrace Violet (10 pM, C34557, Fisher), and cancer cells were labeled with CFSE (10 pM, C34554, Fisher), as described in the manufacturer’s protocol. Macrophages and target cells were then mixed with an E: T ratio of 4: 1, along with the indicated concentrations of antibodies, and incubated at 37 °C and 5% CO2. After 4 hours, adherent cells were detached with accutase, combined with non-adherent cells, and the fluorescence was evaluated by flow cytometry. % phagocytosis was calculated as 100 x (# of double positive cells for CFSE+ and celltrace violet+ cells) / (# of CFSE+ cells).Example 2:Reduced affinity increases the selectivity of activating ADCC reporter cells based on HER2 expression level

[0131] To increase the selectivity of trastuzumab for cells with high HER2 levels, affinity-reduced variants were generated by introducing mutations into the heavy chain CDRs and selecting for mutants with reduced affinity using yeast-surface display. This resulted in the isolation of two variants, namely S4 and S12 (Fig. 1A), which were produced as Fab fragments (Fig. 2) and characterized in terms of their monovalent affinities (Fig. IB). The S4 Fab variant displayed the weakest affinity (KD>150 nM)Docket No. UM-44853.601for high HER2 cells (HCC1954, denoted as HER2+++), and its binding levels did not saturate at the highest tested Fab concentration (600 nM). Tire S 12 Fab variant bound with intermediate affinity (KD of 44.1±6.5 nM) relative to wild type (KD of 9.1±1.2 nM).

[0132] Next, antibodies were reformatted as IgGs, produced, purified (Fig. 2), and evaluated for their binding to cell lines with high (HER2+++) and low (MDA-MB-231, denoted as HER2+) HER2 levels (Fig. 1C and Fig. ID). These cell lines have -1-3 million (HER2+++) and -30,000-40,000 (HER2+) receptors per cell, the latter of which is similar to estimates of -10,000-35,000 HER2 receptors per cell on healthy tissue [32-37], It was observed that binding was similar in all the three IgGs to the HER2+ +cells (EC50 values of 1.4-2.1 nM; Fig. 1C). However, the S4 IgG exhibited significantly weaker binding to the HER2+cells (EC50 >20 nM) relative to the S12 (EC50 of 1.19±0.19 nM) and wild-type (EC50 of 0.75±0.15 nM) antibodies (Fig. ID). It was demonstrated that the bivalent nature of the S4 IgG was sufficient to mediate high avidity binding to HER2+++cells, while its intrinsic affinity played a minimal role in binding to HER2+cells. It was contemplated that this approach finds use for generating trastuzumab mutants with heightened avidity and reduced intrinsic affinity.

[0133] Next, it was then demonstrated that the reduced binding of the S4 IgG to HER2+cells increased selectivity of activating ADCC (FcgRIIIA) reporter cells based on HER2 expression, using trastuzumab and the two affinity -reduced variants (Fig. 3). There was a minimal difference between the three antibody variants in terms of activating FcgRIIIA for HER2+++cells (EC50 values I.06±0.34 nM for WT, 1.59±0.35 nM for S12 and 1.51±0.80 nM for S4), while the S4 IgG was the least potent antibody forthe HER2+cells (EC50 values 0.27±0.1 nM for WT, >2.5 nM for S12 and >10 nM for S4). The ratio of activity as a function of antibody concentration demonstrated that S4 IgG was the most selective for HER2+++cells, especially in the concentration range of 0.13-1.311M. Relative to WT and S12 IgG, S4 IgG displayed significant increases in selectivity for HER+++cells at antibody concentrations of 0.14-1.2 nM (Fig. 4).

[0134] Similar behavior was observed forFc-modified versions of the three trastuzumab variants (Fig.3). Five mutations in the CH2 and CH3 regions, which have been previously reported to increase ADCC activity

[0038] , were introduced into the trastuzumab variants (herein referred to as enhanced Fc1 or eFcl), which showed that the three antibodies displayed similar activity for HER2+++cells (EC50 values 0.37±0.19 nM for WT-eFcl, 0.41±0.16 nM for S12-eFcl and 0.42±0.35 nM for S4-eFc1), while the S4-eFcl IgG was the least potent for HER2+cells (EC50 values 0.09±0.01 nM for WT-eFc1. 0.12±0.02 nM for S12-eFcl and 3.49±1.00 nM for S4-eFcl). Tire ratio of activity as a function of antibody concentration demonstrated that S4-eFc1 IgG was the most selective for HER2+++cells, more specifically in the concentration range of 0.015-0.14 nM. Relative to WT-eFcl and S12-eFcl, S4-eFclDocket No. UM-44853.601displayed statistically significant increases in selectivity for HER2+++cells at antibody concentrations of 0.015-3.7 nM (Fig. 4).

[0135] Additionally, tradeoffs between the maximum activity and selectively for activating ADCC reporter cells based on HER2 expression level were evaluated. For the WT and S12 IgGs, relatively high maximum activities (74-75-fold relative to control), but relatively low maximum selectivities (6.3-7.0 ratios of activity at a given antibody concentration). The eFc1 variants further increased maximum activity (98.0-102.3), but reduced selectivity (2-4-fold). In contrast, the S4 IgG showed a similar maximum activity (68.1±2.9 relative to 74-75 for WT and S12) but with increased selectivity (11.4±0.3 relative to 6.3-7.0 forWT and S12). It was also shown that the S4-eFcl IgG further increased activity (94.7±4.5) without reducing selectivity (11.5±0.6). It was contemplated that S4-eFcl IgG has the best combination of maximum activity and selectivity for activating ADCC reporter cells based on HER2 expression level.Example 3:Mixtures of affinity-reduced and effectorless blocking antibodies maximize ADCC activity and selectivity

[0136] To further improve the selective activation of ADCC reporter cells based on HER2 expression levels, mixtures of two trastuzumab variants were utilized (Fig. 5D). The binding of mixtures of two types of antibodies, namely fluorescently-labeled WT trastuzumab or HALA variants were tested with reduced HER2 affinities in the presence of an unlabeled blocking version of trastuzumab that retains high HER2 affinity (wild-type Fab regions) but was engineered to be effectorless (herein referred to as the “blocker"; Fig. 6). For WT: blocker mixtures, WT trastuzumab binding was progressively reduced to both HER2+++and HER2+cells as the mixture ratio was increased (Fig. 6A-B), resulting in maximum HER2+++ / HER2+binding ratios that were similar with and without the blocking antibody (Fig. 6C). The ratio values were similar to the ratio of expression levels for the two cell lines. However, S12:blocker mixtures resulted in much stronger reduction in binding to HER2+cells (Fig. 6E) relative to HER2+++cells (Fig.6D), resulting in increased binding ratios for the mixtures relative to the single S12 IgG (Fig. 6F). For 1:1 mixtures, the increased binding selectivity was significant at S12 IgG concentrations of -1-10 nM (Fig. 7). In contrast, S4:blocker mixtures results in strong reductions in binding to both HER2+++(Fig. 6G) and HER2+(Fig.6H) cells, resulting in lower binding ratios than those for the S4 IgG alone (Fig. 61).

[0137] After quantifying the binding ratios on cells, the ADCC reporter cell activity for the effector-competent WT, S12, and S4 IgGs were tested in the presence of different ratios of the effectorless blocker (Fig.8). For mixtures with WT trastuzumab IgG, it was found that increasing WTDocket No. UM-44853.601IgG: blocker ratios progressively reduced the maximum activity for HER2+++cells (Fig. 8A).Equimolar (1:1) mixtures result in a maximum activity of 53% relative to the WT IgG alone, while the other mixtures with higher blocker ratios have lower maximum activities (40% for 1:2. 26.7% for 1:4 and 21% for 1:8 antibody: blocker ratio relative to WT IgG alone). A similar trend was observed for the same antibody mixtures on HER2+cells, although the overall activation levels were reduced slightly more than those for HER2+++cells (Fig. 8B). Additionally, WT: blocker ratios of 1:4 and 1:8 reduced maximum activation levels to near baseline values. This resulted in activation ratios that were highest for the 1:4 and 1:8 WT: blocker ratios (Fig. 8C).

[0138] The S12:blocker (Fig. 8D, Fig. 8E and Fig. 8F) and S4:blocker (Fig. 8G, Fig. 8H and Fig. 81) results displayed similar qualitative trends with key quantitative differences relative to the WT: blocker results. First, as observed for the 1:1 WT: blocker ratio (Fig. 8A), the 1:1 S12:blocker (Fig. 8D) and 1:1 S4:blocker (Fig. 8G) ratios resulted in the highest activities for HER2+++cells compared to antibody mixtures at different ratios (i.e. the lowest blocker levels result in the highest activation). It was found that increasing antibody concentrations (even at the same blocker ratio) inhibited activation, including at concentrations >10 nM for S12 mixtures and >3 nM for S4 mixtures. Second, 1:1 S12:blocker (Fig. 8E) and 1:1 S4:blocker (Fig. 8H) mixtures were sufficient to eliminate activity on HER2+cells, which was unique relative to the behavior observed for the WT: blocker mixtures. Third, the low activity of the 1:1 S12:blocker and 1:1 S4:blocker mixtures for HER2+cells lead to higher selectivity, as judged by the HER2+++ / HER2+ratios at each antibody concentration, including maximum values of 36 for S12 mixtures and 30 for S4 mixtures relative to 10.5 for WT mixtures (Fig. 8F and Fig. 81). Fourth, the increased selectivity of S12:blocker mixtures relative to S12 alone was observed at a broad antibody concentration range (statistically significant differences at 0.4-100 nM), while the corresponding S4:blocker mixtures displayed increased selectivity at a smaller range of concentrations (statistically significant differences at 0.3-4 nM; Fig. 9).

[0139] Next, tradeoffs were evaluated between maximal activity and selectivity as a function of HER2 expression level (Fig. 8J, Fig. 8K and Fig. 8L). For the WT: blocker mixtures, the maximum activity was strongly reduced as the antibody ratio was increased, while the selectivity modestly increased and was maximal at the ratios corresponding to the lowest maximal activity (1:8 and 1:4 WT: blocker ratios; Fig. 8). In contrast, the S12:blocker and S4:blocker mixtures displayed maximal selectivities at ratios with intermediate maximal activity (1:1 ratios; Fig. 8K and Fig. 8L). At high S12:blockerand S4:blocker ratios (1:8 and 1:4), the mixtures displayed reduced selectivity along with low maximal activity.

[0140] The antibody mixtures significantly improved the selectivity of effector function relative to a single antibody, but the maximum activity was reduced. To achieve high maximum activity whileDocket No. UM-44853.601maintaining or improving the selectivity of the antibody mixtures for activating ADCC reporter cells based on HER2 expression levels, mixtures were evaluated using antibodies with enhanced Fcregions (Fig. 10). The WT-eFcl antibody displayed ~35% higher maximal activity for HER2+++cells (Fig.10A) relative to the non-enhanced WT antibody (Fig.8A), and this activity was progressively reduced as blocker ratio increased (Fig. 10A). This resulted in -60% reduction in maximum activity at the highest blocker ratio (1:8), which was a smaller reduction than that observed for the corresponding non-enhanced 1:8 WT: blocker mixtures (-80% reduction; Fig. 10A). However, the WT-eFcl antibody also displayed relatively high maximal activity on HER2+cells, and this activity was progressively reduced as the blocker ratio was increased (Fig. 10A). This resulted in -75% reduction in activity at the highest blocker ratio (1:8), which was also a smaller reduction than that observed for the nonenhanced 1:8 WT: blocker mixtures (-85% reduction; Fig. 8A). This modest ability of the blocking antibody to reduce activity on HER2+cells resulted in low selectivity, including a maximal HER2+++ / HER2+activation ratio of 16 for WT-eFc1:blocker mixtures (Fig. 10C).

[0141] Additionally, it was found that the S12-eFcl:blocker and S4-eFcEblocker mixtures displayed distinct patterns of behavior (Fig. 10D, Fig. 10E, Fig. 10F, Fig. 10G, Fig. 10H and Fig. 101). For the S12-eFcEblocker mixtures, increasing antibody ratios led to moderate reduction in maximal activity on HER2+++cells, including a -60% reduction of maximum activity for the highest antibody ratio (1:8;Fig. 10D), which was similar to that observed for the WT-eFc1:blocker mixtures (Fig. 10A). However, the maximum activity with the blocker was similar to the WT antibody alone (Fig. 3). High antibody concentrations progressively reduced activity, as observed for the corresponding non-enhanced mixtures (Fig. 8D), at concentrations >3-10 nM for the different S12-eFcl mixtures (Fig. 10D).Notably, the S12-eFc1:blocker mixtures were unable to fully inhibit activity on HER2+cells except at the highest ratios (1:8 and 1:4; Fig. 10E), which was distinct relative to the non-enhanced S12:blocker mixtures (Fig. 8E). This results in maximal selectivity at the highest antibody ratios for the S12-eFc1:blocker mixtures (1:8 and 1:4; Fig. 10F), which was different than the optimal antibody ratio (1:1) for the non-enhanced S12:blocker mixtures (Fig. 8F). However, even at a suboptimal S12-eFcEblocker ratio of 1:1, it was observed that there were significant increases in selectivity relative to the corresponding selectivity for S12-eFc1 (0.4-100 nM for the 1:1 mixtures; Fig. 11).

[0142] For the S4-eFc1:blocker mixtures, it was observed that there was higher maximal activities for HER2+++cells (Fig. 10G) than that for the corresponding non-enhanced S4:blocker mixtures (Fig.8G). Notably, the S4-eFc1:blocker mixtures did not show activity on HER2+cells (Fig. 10H), as also observed for S4:blocker mixtures (Fig. 8H), which resulted in an increased selectivities for the S4-Docket No. UM-44853.601eFcl:blocker mixtures (Fig. 101) relative to S4:blocker mixtures (Fig. 81). Moreover, significant increases in selectivity of S4-eFcl: blocker mixtures relative to S4-eFcl alone were observed at a relatively broad range of antibody concentrations (0.4-33 nM for 1:1 mixtures; Fig. 11).

[0143] Notably, the tradeoffs between maximal activity and selectivity displayed several distinct patterns of behavior for the enhanced Fcvariants (Fig. 10J, Fig. 10K and Fig. 10L) relative to their wild-type counterparts (Fig. 8J, Fig. 8K and Fig. 8L). For the WT-eFcl:blocker mixtures, the maximum activity was strongly reduced as the antibody: blocker ratio was increased, while the selectivity was largely unchanged (Fig. 10J). In comparison, tire wild-type Fcvariants displayed low er maximal activity with modestly higher selectivity (Fig. 8J). The maximal activity of the S12-eFc1:blocker mixtures was also significantly reduced as the antibody: blocker ratio increased, but the selectivity progressively increased (Fig. 10K). This resulted in the maximum selectivities at the highest S12-eFc1:blocker ratios (1:4 and 1:8, which were not statistically different), while an intermediate antibody ratio was optimal for the S12:blocker mixtures (1:1; Fig. 8K). Notably, the maximum activity (39 for 1:8 S12-eFcl:blocker and 40 for 1:1 S12:blocker) and selectivity (40 for 1:8 S12-eFcl:blocker and 35 for 1:1 S12:blocker) for the two different mixtures was similar. Finally, the maximum activity of the S4-eFcl:blocker mixtures was reduced as the antibody ratio increased, with maximum selectivity at 1:1 and 1:2 antibody ratios (not statistically different; Fig. 10L), which was higher than the corresponding S4:blocker mixtures (Fig. 8L). Importantly, the 1:1 S4-eFc1:blocker mixture resulted in higher maximum activity and selectivity relative to the corresponding nonenhanced mixtures, including an activity of 72-fold induction for 1:1 S4-eFcl:blocker versus 29-fold for 1:1 S4:blocker and a selectivity ratio of 53 for 1:1 S4-eFc1:blocker versus 32 for 1:1 S4:blocker. Overall, the 1:1 S4-eFcl: blocker mixture was able to achieve high activity (72-fold induction; Fig.10L) that was similar to the maximum activation of the ADCC reporter cells for any of the non-Fcenhanced antibodies (71- to 78-fold induction for WT, S12, and S4; Fig. 2) while achieving a 53-fold selectivity compared to the lower (6- to 11-fold) selectivity observed when only modifying the Fab affinity (WT, S12, or S4 alone; Fig. 8).

[0144] To achieve the goal of validating authentic ADCC activity, assessing its applicability across cancer cell lines with varying HER2 expression levels, and evaluating its consistency across different FcyRIIIA allotypes, the following steps were taken: First, it was evaluated whether the findings translated to authentic ADCC activity rather than simply activating FcγRIIIA using ADCC reporter cells. Second, the findings were applied to additional cancer cell lines with varying HER2 expression levels. Third, the consistency of the results across different FcγRIIIA allotypes was examined. First,Docket No. UM-44853.601ADCC activity for S4-eFcl was evaluated in the presence and absence of the blocking antibody (1:1 ratio) using two HER2+++cell lines, namely HCC1954 (~l-3 million receptors per cell) and NCI-N87 (-1-2 million receptors per cell) [35, 39], and two HER2+cell lines, namely MDAMB231 (-30,000-40,000 receptors per cell) and CAPAN1 (-15,000-20,000 receptors per cell) [40, 41], with PBMCs of different allotypes (Fig. 12). Notably, a strong decrease in ADCC activity was observed (i.e., target cell lysis) in the presence of the blocking antibody for both cell lines with low HER2 expression, but not for both cell lines with high HER2 expression (Fig. 12A and Fig. 12B). These results were similar for both FcgRIIIA allotypes (VV and FF at position 158) and resulted in increased HER2 / HER2 lysis ratios (Fig. 12C). Overall, these findings highlight that the S4-eFc1:blocker mixture was able to increase the selectivity of ADCC based on the HER2 expression across different cell lines and allotypes.Example 4:Engineered antibody mixtures also maximize activity and selectivity for antibody-dependent cellular phagocytosis

[0145] It was found that mixtures of engineered antibodies improved ADCC selectivity while maintaining high maximum activity, leading to further evaluation of whether this could also be achieved for a second effector function, namely ADCP. Additionally, it was observed that the effector-competent IgGs (WT, S12 and S4) – in the absence of a blocking antibody – selectively displayed high ADCP reporter cell activity with HER2+++cells but not HER2+cells (Fig. 13). Moreover, it was observed that there were similar levels of ADCP reporter signal onHER2 cells when using equivalent IgGs with eFcl mutations, while also observing a lack of ADCP signal with HER2+cells (Fig. 14) However, when using a different set of previously reported mutations in the Fcregion to enhance effector function

[0042] , which herein are denoted as eFc2, it was observed ~3x greater maximum ADCP signaling activity on HER2+++cells as well as weaker but significant activity on HER2+cells (Fig. 13). For the WT-eFc2 and S12-eFc2 IgGs, this resulted in much lower activation ratios relative to the equivalent antibodies with WT Fcregions. However, the maximum ratio for the S4-eFc2 IgG was larger than the equivalent antibody with a WT Fcregion. The S4-eFc2 IgG displayed statistically higher ADCP receptor selectivity for HER2+++cells at antibody concentrations of 0.015-3.7 nM relative to WT-eFc2 and 0.046-3.7 nM relative to S12-eFc2 (Fig. 15).

[0146] Next, the selectivity of the eFc2 antibodies for activating ADCP reporter cells was tested to determine if it could be enhanced by combining them with an effectorless blocking antibody, similar to the ADCC activity. (Fig. 16). For WT-eFc2:blocker mixtures, the maximum activity was modestly impacted for HER2+++cells (Fig. 16A), but strongly reduced for HER2+cells (Fig. 16B), whichDocket No. UM-44853.601resulted in activation ratios that were much larger than those for WT-eFc2 IgG alone (Fig. 16C). The highest selectivity for the WT-eFc2:blocker mixtures was observed at a ratio of 1:8, which improved the selectivity by 10-fold relative to WT-eFc2 IgG alone while modestly reducing the maximum activity for HER2+++cells by 28%. Moreover, the increased selectivity of WT-eFc2: blocker mixtures relative to WT IgG alone was observed at a broad antibody concentration range (statistically significant differences at 0.1-100 nM for 1:1 mixtures; Fig. 17).

[0147] Tire results for the S12-eFc2 and S4-eFc2 mixtures showed similar qualitative trends but also important differences relative to the WT-eFc2 mixtures (Fig. 16D, Fig. 16E and Fig. 161). Both of the affinity-reduced antibodies displayed similar maximum activation in the presence of a blocking antibody for HF. R2cells - especially at low S12:blocker and S4:blocker ratios (Fig. 16D and Fig.16G) - while largely eliminating activity on HER2+cells (Fig. 16E and Fig. 16H). This results in relatively high maximum activation ratios (Fig. 16F and Fig. 161), especially for S4-eFc2 mixtures. The increased selectivity of S12-eFc2 and S4-eFc2 antibodies in the presence of a blocker was observed at a broad antibody concentration range (statistically significant differences at 0.1-100 nM for 1:1 S12-eFc2:blocker mixtures and 1-100 nM for 1:1 S4-eFc2:blocker mixtures; Fig. 17). A comparison of the maximum activity and selectivity at each IgG:blocker ratio (Fig. 16J, Fig. 16K and Fig. 16L) revealed that the S4-eFc2:blocker mixture at a ratio of 1:2 lead to the optimal combination of high ADCP reporter activity (maximum fold induction of 395 relative to 402 for the S12-eFc2 IgG alone) and selectivity (ratio of 395 relative to 21 for the S12-eFc2 IgG alone). However, the maximal activity and selectivity for the 1:2 S4-eFc2:blocker ratio was not statistically different than the corresponding values for both properties for the 1: 1 and 1:4 ratios, although the maximum activity for the 1:2 S4-eFc2:blocker ratio was statistically larger than that for the 1:4 ratio while the maximum selectivity for the 1:2 ratio was statistically larger than that for the 1: 1 ratio. Overall, the combination of a blocker antibody with the S4-eFc2 antibody was able to increase both the maximum ADCP reporter activity (395-fold) and selectivity (396-fold; Fig. 16) relative to any of the antibodies alone (105-fold activity and 105-fold selectivity; Fig. 13).

[0148] Similar to the ADCC analysis in Fig. 12, the ADCP reporter cells were further evaluated to determine if they translate to authentic ADCP activity, including across different cancer cell lines and FcyRIIA allotypes (Fig. 18). To address these questions, the ADCP activity for S4-eFc2 in the presence and absence of blocker (1:1 ratio) were evaluated using two HER2+++(HCC1954 and NCI-N87) and two HER2+(MDAMB231 and CAPAN1) cell lines for two FcyRIIA allotypes (HH and RR at position 131). Notably, an almost complete blocking in ADCP activity (i.e., target cell phagocytosis) wasDocket No. UM-44853.601observed in the presence of the blocking antibody for both cell lines with low HER2 expression, while little blocking for both cell lines were observed with high HER2 expression (Fig. 18A and Fig. 18B).These findings indicated that the S4-eFc2:blocker mixture increased the selectivity of ADCP based on HER2 expression levels across different cell lines and FcyRIIA allotypes.REFERENCES

[0149] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. 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Mazor, Y., et al., Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence. Sci Rep, 2017. 7: p. 40098.28. Ho, S. K., etal., Epitope and Fc-Mediated Cross-linking, but Not High Affinity, Are Critical for Antitumor Activity of CD 137 Agonist Antibody with Reduced Liver Toxicity. Mol Cancer Ther, 2020. 19(4): p. 1040-1051.29. Liu, X., et al., A cross-reactive pH-dependent EGFR antibody with improved tumor selectivity and penetration obtained by structure -guided engineering. Mol Ther Oncolytics, 2022. 27: p. 256-269.30. Sulea, T., et al., Structure-based engineering of pH-dependent antibody binding for selective targeting of solid-tumor microenvironment. MAbs, 2020. 12(1): p. 1682866.31. Liu, Y., et al., An antibody Fc engineered for conditional antibody-dependent cellular cytotoxicity at the low tumor microenvironment pH. J Biol Chem, 2022. 298(4): p. 101798.32. Kliera, E., et al., Cellular-Resolution Imaging of Bystander Payload Tissue Penetration from Antibody-Drug Conjugates. Mol Cancer Ther, 2022. 21(2): p. 310-321.33. Hendriks, B. S., et al.. Impact of tumor HER2 / ERBB2 expression level on HER2 -targeted liposomal doxorubicin-mediated drug delivery: multiple low-affinity interactions lead to a threshold effect. Mol Cancer Ther, 2013. 12(9): p. 1816-28.34. Costantini, D. L., et al., Trastuzumab-resistant breast cancer cells remain sensitive to the auger electron-emitting radiotherapeutic agent HlIn-NLS-trastuzumab and are radiosensitized by methotrexate. J Nucl Med, 2008. 49(9): p. 1498-505.35. Major, M., et al., Surface-Available HER2 Levels Alone Are Not Indicative of Cell Response to HER2 -Targeted Antibody-Drug Conjugate Therapies. Pharmaceutics, 2024. 16(6).Docket No. UM-44853.60136. Onsum, M. D., et al., Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2 -positive patients. Am J Pathol, 2013. 183(5): p. 1446-1460.37. Moutafi, M., et al., Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer. Lab Invest, 2022. 102(10): p. 1101-1108.38. Stavenhagen, J. B., et al., Fc optimization of therapeutic antibodies enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcgamma receptors. Cancer Res, 2007. 67(18): p. 8882-90.39. Li, J. Y., et al., A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2 -Targeted Therapy. Cancer Cell, 2016. 29(1): p. 117-29.40. Larbouret, C., et al., In pancreatic carcinoma, dual EGFR / HER2 targeting with cctuximab / trastuzumab is more effective than treatment with trastuzumab / crlotinib or lapatinib alone: implication of receptors' down-regulation and dimers' disruption. Neoplasia, 2012. 14(2): p. 121-30.41. Gaborit. N.. et al., Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR / HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies. J Biol Chem, 2011. 286(13): p. 11337-45.42. Richards, J. O., et al., Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther, 2008. 7(8): p. 2517-27.43. Bordeau, B. M., Y. Yang, and J. P. Balthasar, Transient Competitive Inhibition Bypasses the Binding Site Barrier to Improve Tumor Penetration of Trastuzumab and Enhance T-DM1 Efficacy. Cancer Res, 2021. 81(15): p. 4145-4154.44. 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[0150] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims

Claims

CLAIMSWhat is claimed is:

1. A therapeutic composition comprising a first antibody and a second antibody that each bind to a cellular target molecule, wherein the first antibody, compared to the second antibody, has a 10-fold or more lower affinity for the cellular target molecule.

2. A kit comprising a first therapeutic composition comprising a first antibody that binds to a cellular target molecule and a second therapeutic composition comprising a second antibody that binds to the cellular target molecule, wherein the first antibody, compared to the second antibody, has a 10-fold or more lower affinity for the cellular target molecule3. A method comprising: administering to a subject a first antibody and a second antibody that each bind to a cellular target molecule, wherein the first antibody, compared to the second antibody, has a 10-fold or more lower affinity for the cellular target molecule.

4. The method of claim 3, wherein said first antibody and second antibody are administered simultaneously to the subject.

5. The method of claim 3, wherein said first antibody and second antibody are administered sequentially to the subject.

6. The method of claim 5, wherein the second antibody is administered before the first antibody.

7. The method of claim 5, wherein the first antibody is administered before the second antibody.

8. The composition, kit, or method of any of claims 1-7, wherein the second antibody has reduced effector function relative to the first antibody.

9. The composition, kit, or method of any of claims 1-8, wherein the second antibody has no effector function.

10. The composition, kit, or method of claim 9, wherein the second antibody has no Fcdomain.

11. The composition, kit, or method of any of claims 1-9, wherein the second antibody comprises an Fcdomain comprising one or more variant amino acid sequences or modifications that reduce effector function.

12. The composition, kit, or method of any of claim 11, wherein effector function is reduced by one or more of the following modifications: (i) introduction of specific amino acid mutations in the Fcregion, (ii) modification of Fcglycosylation patterns, (iii) deletion or mutation of Fcy receptor binding sites or, (iv) truncation or removal of tire Fcregion.

13. The composition, kit, or method of any of claim 11, wherein the Fcregion comprises SEQ IDN0s:8.

14. The composition, kit, or method of any of claims 1-13, wherein the cellular target molecule comprises a cellular receptor.

15. The composition, kit, or method of any of claims 1-14, wherein the target molecule is overexpressed on cancer cells.

16. The composition, kit, or method of any of claims 1-15, wherein the first antibody is a variant of the second antibody.

17. The composition, kit, or method of 16, wherein the first antibody comprises a variant Fcregion that has enhanced effector function.

18. The composition, kit, or method of 17, wherein the Fcregion comprises SEQ ID NOs: 6 or 7.

19. The composition, kit, or method of any of claims 1-18, wherein the first antibody, compared to the second antibody, has a 25 -fold or more lower affinity for the cellular target molecule.

20. The composition, kit, or method of any of claims 1-19, wherein the first antibody, compared to the second antibody, has a 100-fold or more lower affinity for the cellular target molecule.

21. The composition, kit, or method of any of claims 1-20, wherein the second antibody has reduced affinity for an Fcreceptor compared to said first antibody.

22. The composition, kit, or method of claim 21, wherein the Fcreceptor is one or more of FcγRI, FcγRII, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIII, FcγRIIIa or FcγRIIIb.

23. The composition, kit, or method of any of claims 1 -22, wherein the cellular target molecule is human epidermal growth factor receptor 2 (HER2).

24. The composition, kit, or method of claim 21. wherein the first and second antibodies are variants of Trastuzumab.

25. The composition, kit, or method of claim 24, wherein the first antibody comprises SEQ ID Nos: 3 or 4.

26. Use of a composition or kit of any of claims 1-2 or 8-25.

27. Use of a composition or kit of any of claims 1-2 or 8-25 to treat a disease or condition having disease cells that overexpress said cellular target molecule.

28. The use of claim 27, wherein antibody-dependent cellular cytotoxicity (ADCC) selectivity and / or antibody-dependent cellular phagocytosis (ADCP) selectivity are altered relative to a treatment with the first antibody alone or treatment with an antibody having the affinity of the second antibody and the effector function of the first antibody, or similar effector function.

29. Use of a composition or kit of any of claims 1-2 or 8-25 for the treatment of cancer.

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