Therapeutic uses of EGFR targeted bispecific polypeptides

Abstract

Provided are compositions and methods for treating cancers, in particular those characterized with EGFR expression. The compositions include a conditionally-activatable XTENylated Protease-Activated bispecific T Cell Engager targeting EGFR (EGFR-XPAT) that remain inactive until activated by proteases present in tumor microenvironment. The EGFR-XPAT exhibited exceptional safety profiles even at high doses and were efficacious in cancer patients resistant to even multiple lines of prior therapies.

Description

Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO THERAPEUTIC USES OF EGFR TARGETED BISPECIFIC POLYPEPTIDES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the United States Provisional Application Serial Nos. 63 / 779,752, filed March 28, 2025, and 63 / 815,363, filed May 30, 2025, the content of each of which is hereby incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (4O2128.xml; Size: 17,772 bytes; and Date of Creation: March 16, 2026) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Epidermal growth factor receptor (EGFR) is one of the most well-characterized oncogenes in human cancer and promotes tumorigenesis through the activation of downstream signal transduction pathways. Approved EGFR-directed therapies have been successful at improving progression-free survival (PFS) and overall survival (OS) in certain patient populations across different tumor types (e.g.. non-small-cell lung cancer [NSCLCI with activating EGFR mutations, colorectal cancer [CRC] without KRAS mutations, head and neck squamous cell carcinoma [HNSCC]); however, most patients ultimately become refractory to treatment as a result of innate or acquired resistance. Despite EGFR overexpression in other solid tumor indications (e.g., pancreatic ductal adenocarcinoma [PDAC], renal cell carcinoma (RCC), CRC [RAS mutant]), EGFR-directed therapies that block EGFR signaling have achieved limited success. Thus, patients whose tumors are anti-EGFR-refractory or tumor types for which existing EGFR therapies have not shown efficacy represent an unmet need, and novel effective treatments are warranted.

[0004] Bispecific T Cell Engagers (TCEs) direct T cell cytotoxicity to tumors that express a selected tumor associated antigen, bypassing the requirements for T cell recognition of tumor antigens. The activity of a TCE depends on its ability to activate T cells through effective stimulation of the T cell receptor (TCR). Their extreme potency derives from the minimal requirement for as few as three TCRs to become stimulated and coalesce to form an immune synapse between the T cell and target cell to initiate cytotoxicity. In addition to their induction of cytotoxicity, their potency also involves cytokine driven actions downstream ofVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO T cell activation that enhance and amplify the antitumor immune response. Thus, TCEs offer the promise of immunotherapy to patients whose tumors harbor insufficient mutations or have escaped immune surveillance by other means.

[0005] TCEs’ use has been limited by on-target toxicities against normal tissue expressing even low levels of the target antigen. Toxicities such as cytokine release syndrome at low doses have prevented dose escalation to reveal the modality’s clinical potential. For instance, grade 4 cytokine release syndrome induced in patients treated with TCRs highlights the challenge faced by TCEs even when directed against relatively tissue-restricted targets.

[0006] Attempts to circumvent CRS include complex molecular designs, but these have been unsuccessful due to toxicity and / or enhanced immunogenicity. This presents a significant unmet need for new strategies that can overcome therapeutic index challenges in solid tumors. If the potency of TCEs could be harnessed and the CRS and on-target toxicity challenges could be controlled, it may be possible to generate powerful therapeutics that could potentially be used against a broad spectrum of cancers.SUMMARY

[0007] The present disclosure provides compositions and methods for treating cancers, in particular those characterized with EGFR expression. The compositions include a conditionally-activatable XTENylated Protease- Activated bispecific T Cell Engager targeting EGFR (EGFR-XPAT) that remain inactive until activated by proteases present in tumor microenvironment. The example EGFR-XPAT, referred to as XPAT01, exhibited exceptional safety profiles in animal studies.

[0008] In accordance with one embodiment of the present disclosure, provided is a method for treating cancer in a patient in need thereof, comprising administering to the patient a dose of at least 1 pg / kg of a polypeptide once every week to once every eight weeks, wherein the polypeptide comprises (a) a first extended recombinant polypeptide (XTEN) having at least 90% sequence identity to SEQ ID NO:2, (b) a core fragment comprising an anti-EGFR antigen-binding fragment and an anti-CD3 antigen-binding fragment, and (c) a second XTEN having at least 90% sequence identity to SEQ ID NO: 12, and wherein (a) is connected to (b) through a first protease-cleavable site, and (b) is connected to (c) through a second protease-cleavable site.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0009] Also provided, in one embodiment, is a method for enhancing immune cell infiltration into a solid tumor in a cancer patient, comprising administering to the patient a dose of at least 1 g / kg of a polypeptide once every week to once every eight weeks, wherein the polypeptide comprises (a) a first extended recombinant polypeptide (XTEN) having at least 90% sequence identity to SEQ ID NO:2, (b) a core fragment comprising an anti-EGFR antigen-binding fragment and an anti-CD3 antigen-binding fragment, and (c) a second XTEN having at least 90% sequence identity to SEQ ID NO: 12, and wherein (a) is connected to (b) through a first protease-cleavable site, and (b) is connected to (c) through a second protease-cleavable site.

[0010] Further provided is a method for treating cancer in a patient in need thereof, comprising administering to the patient a dose of at least 1 pg / kg of a polypeptide once every week to once every eight weeks, wherein the patient is being treated with an anti-PD-1 or anti-PD-Ll inhibitor, wherein the polypeptide comprises (a) a first extended recombinant polypeptide (XTEN) having at least 90% sequence identity to SEQ ID NO:2, (b) a core fragment comprising an anti-EGFR antigen-binding fragment and an anti-CD3 antigenbinding fragment, and (c) a second XTEN having at least 90% sequence identity to SEQ ID NO: 12, and wherein (a) is connected to (b) through a first protease-cleavable site, and (b) is connected to (c) through a second protease-cleavable site.

[0011] Yet another embodiment provides a method for enhancing immune cell infiltration into a solid tumor in a cancer patient, comprising administering to the patient a dose of at least 1 pg / kg of a polypeptide once every week to once every four weeks, wherein the patient is being treated with an anti-PD-1 or anti-PD-Ll inhibitor, wherein the polypeptide comprises (a) a first extended recombinant polypeptide (XTEN) having at least 90% sequence identity to SEQ ID NO:2, (b) a core fragment comprising an anti-EGFR antigen-binding fragment and an anti-CD3 antigen-binding fragment, and (c) a second XTEN having at least 90% sequence identity to SEQ ID NO: 12, and wherein (a) is connected to (b) through a first protease-cleavable site, and (b) is connected to (c) through a second protease-cleavable site.

[0012] In some embodiments, the anti-PD-1 or anti-PD-Ll inhibitor is an anti-PD-1 or anti-PD-Ll antibody. In some embodiments, the anti-PD-1 or anti-PD-Ll inhibitor is pembrolizumab. In some embodiments, the pembrolizumab is administered at 200 mg once every three weeks, or 400 mg once every six weeks.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0013] In some embodiments, the cancer is characterized with PD-L1 expression.

[0014] In some embodiments, the patient is administered 3 pg / kg to 800 pg / kg of the polypeptide once every week to once every four weeks. In some embodiments, the patient is administered 30 pg / kg to 500 pg / kg of the polypeptide once every week to once every four weeks.

[0015] In some embodiments, each dose of the polypeptide is between 0.2 mg and 200 mg.12. In some embodiments, each dose is 0.2 mg, 0.5 mg, 0.75 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg.

[0016] In some embodiments, the administration results in increase of GM-CSF, IFN-y, IL-ip, IL-2, IL-4, IL-6, IL-10, MCP-1, or TNF-a.

[0017] In some embodiments, the cancer is characterized with EGFR expression. In some embodiments, the cancer is characterized with EGFR immunohistochemistry (IHC) grade 2+ or 3+, in situ hybridization positive (ISH+), or an activating EGFR mutation.

[0018] In some embodiments, the cancer is selected from the group consisting of breast cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, ovarian cancer, bladder cancer, colorectal cancer, endometrial cancer, head and neck cancer, lung cancer and salivary gland cancer.

[0019] In some embodiments, the cancer is colorectal cancer (CRC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinomas (HNSCC), cutaneous squamous cell carcinoma (CSCC), pancreatic ductal adenocarcinoma (PDAC) and renal cell carcinoma (RCC). Tn some embodiments, the cancer patient has metastatic non-small cell lung cancer (NSCLC) or metastatic colorectal cancer (CRC) and has progressed on a prior treatment. In some embodiments, the CRC is characterized as microsatellite stable (MSS). In some embodiments, the cancer patient is resistant to or has progressed from one or more lines of prior treatments. In some embodiments, the one or more lines of prior treatments comprise an anti-EGFR therapy. In some embodiments, the anti-EGFR therapy comprises one or more of gefitinib, erlotinib, afatinib, brigatinib, icotinib, cetuximab, osimertinib, panitumumab, zalutumumab, nimotuzumab, and matuzumab.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0020] In some embodiments, the method further comprises administering to the patient a steroid. In some embodiments, the steroid is administered prior to the first administration of the polypeptide. In some embodiments, the steroid is administered after detection of a cytokine release syndrome (CRS) or pneumonitis in the patient. In some embodiments, detection of the CRS comprises detection of an increase of IL-6 expression in the patient. In some embodiments, the steroid is dexamethasone, and the dexamethasone is administered for no more than 20 mg every 6 hours or longer. In some embodiments, the patient is not treated with a steroid during administration of the polypeptide or is not pre-treated with a steroid.

[0021] In some embodiments, the anti-EGFR antigen-binding fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:5.

[0022] In some embodiments, the anti-CD3 antigen-binding fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the first and second protease-cleavable sites each, independently, has at least 90% sequence identity to SEQ ID NOG.

[0023] In some embodiments, the polypeptide has at least 90% sequence identity to SEQ ID NO:1. In some embodiments, polypeptide comprises the amino acid sequence of SEQ ID NO:1.

[0024] Also provided, in one embodiments, is a method for treating a solid tumor in a patient in need thereof, comprising administering to the patient a dose of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 once every one, two, three, four, five, or six weeks.

[0025] In some embodiments, the method further comprises administering to the patient a first step-up dose of the polypeptide before the dose, wherein the first step-up dose is less than the dose.

[0026] In some embodiments, the method further comprises administering to the patient a second step-up dose of the polypeptide after the first step-up dose and before the dose, wherein the second step-up dose is less than the dose and greater than the first step-up dose.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0027] In some embodiments, the method further comprises administering to the patient a third step-up dose of the polypeptide after the second step-up dose and before the dose, wherein the third step-up dose is less than the dose and greater than the second step-up dose.

[0028] In some embodiments, the dose is between 0.2 mg and 200 mg. In some embodiments, the dose is 0.2 mg, 0.5 mg, 0.75 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg.

[0029] In some embodiments, the polypeptide is administered to the patient intravenously or subcutaneously.

[0030] In some embodiments, the method further comprises administering to the patient an additional therapeutic agent. In some embodiments, the additional therapeutic agent in administered to the patient intravenously or subcutaneously. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor, a tyrosine kinase inhibitor (TKI), a VEGF / VEGFR inhibitor, a RAS pathway inhibitor, a MET inhibitor, a PARP inhibitor, a CDK4 / 6 inhibitor, a bispecific antibody, a chemotherapy, or a radiotherapy. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, and tislelizumab. In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0031] In some embodiments, the method further comprises administering to the patient: (i) 200 mg of pembrolizumab once every three weeks, or (ii) 400 mg of pembrolizumab once every six weeks.

[0032] In some embodiments, the method comprises administering to the patient: (i) a dose of the polypeptide once every week, and (ii) 200 mg of pembrolizumab once every three weeks. In some embodiments, the method comprises administering to the patient: (i) a dose of the polypeptide once every two weeks, and (ii) 400 mg of pembrolizumab once every six weeks. In some embodiments, the method comprises administering to the patient: (i) a dose of the polypeptide once every three weeks, and (ii) 200 mg of pembrolizumab once every three weeks.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0033] In some embodiments, the method further comprises the patient receiving a premedication prior to the polypeptide. In some embodiments, the pre-medication is selected from the group consisting of an antihistamine, an antipyretic, a steroid, an IL-6 receptor antagonist, and any combination thereof.

[0034] In some embodiments, the solid tumor expresses epidermal growth factor receptor (EGFR). In some embodiments, the solid tumor is a lung cancer, prostate cancer, breast cancer, colorectal cancer (CRC), head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, pancreatic cancer, or skin cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the head and neck cancer is head and neck squamous cell carcinomas (HNSCC). In some embodiments, the skin cancer is cutaneous squamous cell carcinoma (CSCC).

[0035] In some embodiments, the solid tumor is a metastatic solid tumor. In some embodiments, the solid tumor is characterized by EGFR gene amplification. In some embodiments, the patient has received a prior cancer therapy. In some embodiments, the patient is refractory to the prior cancer therapy. In some embodiments, the prior cancer therapy is an EGFR-directed therapy. In some embodiments, the prior administered EGFR-directed therapy is a tyrosine kinase inhibitor (TKI) or an anti-EGFR antibody.

[0036] In some embodiments, the patient expresses a biomarker predictive of a response to the polypeptide.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 shows in vitro T-cell-dependent cellular cytotoxicity of XPAT01 and its metabolites against EGFR-cxprcssing human cancer cell lines.

[0038] FIG.2 shows in vitro T-cell-dependent cellular cytotoxicity of XPAT01 and its metabolites against normal primary human keratinocytes.

[0039] FIG.3 shows quantification of relative XPAT01-TCE after 48-hour incubation of 1 or 10 nM XPAT01 with 4 independent cell lines.

[0040] FIG.4 shows in vitro cytokine secretion by XPAT01 and its metabolites following incubation with human PBMCs and HT-29-Luc2 cells.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0041] FIG.5 shows In vitro T-cell activation by XPAT01 and its metabolites with HT-29-Luc2 cells.

[0042] FIG.6 shows the results of efficacy evaluation of XPAT01, XPATOl-NoClvSite, and XPATOl-TCEin human PBMC-engrafted NSG mice bearing LoVo CRC tumors. NSG mice were inoculated subcutaneously with 5 x 1()6 LoVo tumor cells (Day 0), engrafted with 1 x 1()7 PBMCs (Day 5), and treated with the indicated test articles on days denoted by the arrows.

[0043] FIG.7 shows evaluation results of CD8+ cells in human PBMC-engrafted NSG mice bearing LoVo CRC tumors following treatment with XPAT01.

[0044] FIG.8 shows the results of efficacy evaluation of XPAT01, XPAT01-TCE, and XPATOl-NoClvSite in human PBMC-engrafted NSG mice bearing HT-29 CRC tumors. NSG mice were inoculated subcutaneously with 5 x 106HT-29 tumor cells (Day 0), engrafted with 1 x 107PBMCs (Day 0), and treated with the indicated test articles on days denoted by the arrows.

[0045] FIG. 9 shows the results of efficacy evaluation of XPAT01 and XPAT01-TCE in human PBMC-engrafted NSG mice bearing MDA-MB-231 breast cancer tumors. NSG mice were inoculated subcutaneously with 5 x 106MDA-MB-231 tumor cells (Day 0) engrafted with 1 x 107PBMCs (Day 8) and treated with the indicated test articles on days denoted by the arrows.

[0046] FIG. 10A-B show the results of efficacy evaluation of XPAT01 and pembrolizumab in human PBMC-engrafted NSG mice bearing SK-OV-3 ovarian cancer tumors. NSG mice were inoculated subcutaneously with 5 x 106SK-OV-3 tumor cells (Day 0), engrafted with 1 x 107PBMCs (Day 18), and treated with the indicated test articles on days denoted by the arrows.

[0047] FIG. 11 shows individual animal XPAT01 plasma concentration-over- time profiles following a single 30-minute IV infusion of XPAT01 in male cynomolgus monkeys.

[0048] FIG. 12 shows individual animal XPAT01 plasma concentration-over-time profiles following repeated intravenous administration in male and female cynomolgus monkeys.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0049] FIG. 13 shows mean XPAT01 plasma concentration-over-time profiles following repeated intravenous administration in male and female cynomolgus monkeys. Based on a mean of 5 animals per dose group. XPAT01 mean concentrations reported as below the assay LLOQ are displayed at one-half LLOQ.

[0050] FIG. 14A-B show the clearance (A) and cytokine induction (B) following non-clinical administration of XPAT01 and XPAT01-TCE at different doses.

[0051] FIG. 15 provides a graphic description of certain study designs for XPAT01.Abbreviations: BOIN, Bayesian optimal interval; BRAFwt, wild-type BRAF; CRC, colorectal cancer; DL, dose level; EGFR, epidermal growth factor receptor; HNSCC, head and neck squamous cell carcinoma; MAD, maximum administered dose; MSS, microsatellite-stable; NSCLC, non-small-cell lung cancer; Pembro, pembrolizumab; Q3W, every 3 weeks; QW, once weekly.

[0052] FIG. 16A-C illustrate certain dosing regimens for XPAT01, alone or in combination with other therapeutic agents. Abbreviations: D, Day; DLT, dose-limiting toxicity; Q3W, every 3 weeks; QW, once weekly. Note: Each cycle is 21 days, a: Optional step-up dosing may be enabled based on emerging data to optimize the XPAT01 safety profile.DETAILED DESCRIPTIONDefinitions

[0053] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0054] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The termVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0055] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present disclosure.

[0056] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 %) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.

[0057] The term “an equivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucleotide sequence having a certain degree of homology, or sequence identity, with the nucleotide sequence of the nucleic acid or complement thereof. A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof. Likewise, “an equivalent polypeptide” refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four or five addition, deletion, substitution and their combinations thereof as compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequenceVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO retains the activity (e.g., epitope-binding) or structure (e.g., salt-bridge) of the reference sequence.

[0058] As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but arc not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0059] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0060] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.

[0061] The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (y, p, a, 5, E) with some subclasses among them (e.g., yl- y4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGi, IgCi2, IgGs, IgG4, IgGs, etc. are well characterized and are known to confer functionalVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.

[0062] In naturally occurring antibodies, the six “complementarity determining regions” or “CDRs” present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, referred to as “framework” regions, show less inter-molecular variability. The framework regions largely adopt a P-sheet conformation and the CDRs fomi loops which connect, and in some cases form part of, the P -sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0063] In the case where there are two or more definitions of a term which is used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO This particular region has been described by Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest’’ (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0064] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0065] By “specifically binds” or “has specificity to,” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”

[0066] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms,Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0067] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

[0068] As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and / or for treatment.

[0069] A “vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and / or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

[0070] The term “ti / 2” as used herein means the terminal half-life calculated as ln(2) / Kei . Kei is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve. Half-life typically refers to the time required for half the quantity of an administered substance deposited in a living organism to beVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO metabolized or eliminated by normal biological processes. The terms “ti / 2”, “terminal halflife”, “elimination half-life” and “circulating half-life” are used interchangeably herein.

[0071] A “therapeutic effect,” as used herein, refers to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or prevention of disease condition in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, caused by a fusion polypeptide of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0072] The terms “therapeutically effective amount” and “therapeutically effective dose,” as used herein, refers to an amount of a biologically active protein, either alone or as a part of a fusion protein composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. The disease condition can refer to a disorder or a disease.

[0073] The term “therapeutically effective dose regimen,” as used herein, refers to a schedule for consecutively administered doses of a biologically active protein, either alone or as a part of a fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition.Therapeutic Uses of EGFR Targeted Bispecific XPAT

[0074] While TCEs have been shown to be effective in inducing remission in certain cancers, they have not produced widespread therapeutics due to on target, off tumor toxicitics in healthy tissues. By way of explanation, the TCEs form a bridge between T cells and tumor cells and activate T cell-mediated of the tumor cell and further initiating a cytokine amplification cascades that promotes further killing and potentially provides long term immunity. T cells activated by TCEs to release cytolytic perforin / granzymes in a manner that is independent of antigen-MHC recognition. This creates a two-fold response: direct tumor cell death and amplification of tumor killing through initiation of a powerful cytokine response from the tumor cells. The direct tumor cell death results in release of tumorVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO antigens. The cytokine response includes, among others, increased interferon-y which stimulates CD8 T cell activity and stimulates antigen presentation by APCs; increased 1L2 which causes increased proliferation of activated T-cells, and increased CXCL9 and 10 response which increases T cell recruitment. Together the release of tumor antigens and the initiation of the cytokine response results in activation of the endogenous T-cell response which potentially cases epitope spreading to induce long term immunity.

[0075] The toxicity challenge with TCEs arises out the fact that most tumor targets are, to some extent, also expressed in healthy tissue, and normal cells also can produce the cytokines response resulting in cytokine release syndrome (CRS). These two powerful responses of health tissue to T cell activation by TCEs results in an overall lack therapeutic index for these agents.

[0076] The present disclosure has tested a conditionally-activated TCE, XTENylated Protease- ctivated bispecific T Cell Engager targeting EGFR (referred to herein as EGFR-XPAT, and exemplified as XPAT01). XPAT01 (SEQ ID NO:1) is capable of exploiting the dysregulated protease activity present in tumors vs. healthy tissues, enabling expansion of the therapeutic index.

[0077] The active core of XPAT01 includes two single chain antibody fragments (scFvs) targeting CD3 and EGFR, respectively. Two unstructured polypeptide masks (XTENs) are attached to the core that sterically reduce target engagement of EGFR and / or CD3 and extend protein half-life. Such properties of XPAT01 also minimize the potential for immunogenicity, as its lack of stable tertiary structure disfavors antibody binding and the absence of hydrophobic, aromatic and positively charged residues that serve as anchor residues for peptide MHC II binding reduces the potential for T cell epitopes.

[0078] Each XTEN is connected to the core through a protease cleavable site that is recognized by one or more proteases present in tumor tissues. These protease cleavage sites enable proteolytic activation of XPAT01 in the tumor microenvironment, unleashing a small, highly potent TCE that is capable redirecting cytotoxic T cells to kill target-expressing tumor cells. In healthy tissues, where protease activity is tightly regulated, XPAT01 can remain predominantly inactive as intact prodrugs, thus expanding the therapeutic index compared to unmasked TCEs.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0079] The protease cleavable sites used in XPAT01 can be cleaved across a broad array of tumors by proteases (e.g., MMP-2, MMP-7, MMP-9, MMP-13, MMP-14, urokinase (uPA), and matriptase) that are collectively involved in each cancer hallmark (growth; survival and death; angiogenesis; invasion and metastasis; inflammation; and immune evasion). Thus, TCE activity of XPAT01 can be localized to tumors by exploiting the enhanced protease activity that is upregulated in different stages of cancer and tumor development but is tightly regulated in healthy tissues.

[0080] The components of XPAT01 have been optimized to achieve the desired balance between providing sufficient protection in healthy tissue while retaining the necessary potency in tumors across a broad range of cancers. To reduce the potential for T cell activation by the prodrag, a lower binding affinity was selected for the anti-CD3 scFv in addition to a longer XTEN mask (582 amino acid mask (SEQ ID NO: 12) vs. 294 amino acids (SEQ ID NO:2) on the anti-EGFR side). To ensure sufficient activation of XPAT01 in the tumor, the protease release site at the base of the XTEN masks was engineered to be cleaved by at least different proteases among three different classes over-expressed or dysregulated in cancer; these include several matrix metalloproteinases (MMPs), Matriptase, and uPA. As a safety checkpoint, co-engagement of both CD3 and EGFR by XPAT01 is required for T cell activation. Activation of T cells should not occur if XPAT01 is unmasked in inflamed tissues where EGFR expression is absent or if it encounters EGFR expressed in healthy tissue where proteases are tightly controlled. This AND-gate feature is hereby shown to provide preferential activation in the tumor where both elevated protease activity and high EGFR expression are present.

[0081] The presence of the XTEN on XPAT01 produces a prodrag with a long half-life, weak target engagement and negligible T-cell activation. Once the XTEN is removed by the action of the proteases in the tumor microenvironment, this preferential activation of XPAT01 produces an activated TCE (XPAT01-TCE) that has a short half-life, optimal target engagement, and highly efficient T-cell activation, thereby producing a powerful activated drag with an enhanced therapeutic index.

[0082] As demonstrated in in vivo studies (Example 1), XPAT01 remained predominantly inactive as a full masked protein and was cleaved by proteases at the tumor sites to release its active core, XPAT01-TCE, that mediated highly potent, target-directed cytotoxicity, T-cell activation, and cytokine secretion at low picomolar concentrations.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0083] As noted, a major potential adverse event commonly associated with TCE is cytokine release syndrome (CRS). The treatments with XPAT01, it is contemplated, would only result in CRS of low grades; hence no prophylactic steroid treatment is needed. Even if steroid is used, it is contemplated, relatively lower doses of a steroid would be sufficient. Such properties, therefore, underscore the magnificent safety profiles of XPATO 1.

[0084] XPATO 1 is effective in treating EGFR-expressing cancers. Responses to the XPAT01 treatment can be evaluated with methods tested herein. For instance, tumor size changes can be measured with CT scan. Biomarkers can also be used for this purpose, such as the increase of IFNy or decrease of circulating tumor DNA (ctDNA). Interestingly, certain biomarkers, such as activation / secretion of GM-CSF, IFN-y, IL- 10, IL-2, IL-4, IL-6, IL- 10, MCP-1, or TNF-a, may precede tumor shrinkage and thus can serve as prognosis markers.

[0085] Finally, pharmacokinetic / pharmacodynamics results show that XPATO 1 has long half life which supports once weekly, once every two weeks, once every three weeks, or even once every four, six, eight or sixteen weeks dosing.

[0086] In accordance with one embodiment of the present disclosure, therefore, provided is a method for treating cancer in a patient in need thereof. In some embodiments, the cancer is a solid tumor. In some embodiments, the method entails administering to the patient a polypeptide of the instant disclosure, such as XPATO 1 and variants, analogs and derivatives thereof, which are described in further details below. Such polypeptides, containing an activatable TCE (T-cell engager) core having specificity to EGFR and CD3, are also referred to as XTENylated Protease- Activated bispecific T Cell Engager targeting EGFR (EGFR-XPAT).

[0087] Also provided, in one embodiment, is a method for enhancing immune cell infiltration into a solid tumor in a cancer patient, which entails administering to the patient a polypeptide of the instant disclosure, such as EGFR-XPAT.

[0088] Combination therapies have also been tested for EGFR-XPAT. One embodiment provides a method for treating cancer (or enhancing immune cell infiltration into a solid tumor, or improving the efficacy of a checkpoint inhibitor therapy) in a patient in need thereof, wherein the method entails administering to the patient a polypeptide of the instant disclosure (e.g., EGFR-XPAT) and an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anti-PD-1 or anti-PD-Ll inhibitor. Another embodimentVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO provides a method for treating cancer in a patient in need thereof, wherein the method entails administering to the patient a polypeptide of the instant disclosure (e.g., EGFR-XPAT) and the patient is further treated with an anti-PD-1 or anti-PD-Ll inhibitor. Another embodiment provides a method for treating cancer in a patient in need thereof, wherein the method entails administering to the patient an anti-PD-1 or anti-PD-Ll inhibitor wherein the patient is further treated with a polypeptide of the instant disclosure (e.g., EGFR-XPAT).

[0089] A PD-1 inhibitor is a molecule that binds to and inhibits the biological activity of the PD-1 protein. Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a protein on the surface of cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. Examples are anti-PD-1 antibodies and fragments thereof, such as those described below.

[0090] Pembrolizumab (formerly MK-3475 or lambrolizumab, Keytruda) is an anti-PD-1 monoclonal antibody developed by Merck and first approved by the Food and Drag Administration in 2014 for the treatment of melanoma. It was later approved for metastatic non-small cell lung cancer and head and neck squamous cell carcinoma.

[0091] Nivolumab (Opdivo) is an anti-PD-1 monoclonal antibody developed by Bristol-Myers Squibb and first approved by the FDA in 2014 for the treatment of melanoma. It was later approved for squamous cell lung cancer, renal cell carcinoma, and Hodgkin’ s lymphoma.

[0092] Cemiplimab (Libtayo) is an anti-PD-1 monoclonal antibody developed by Regeneron Pharmaceuticals and first approved by the FDA in 2018 for the treatment of cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or curative radiation.

[0093] Spartalizumab (PDR001) is an anti-PD-1 monoclonal antibody developed by Novartis to treat both solid tumors and lymphomas.

[0094] Camrelizumab (SHR1210) is an anti-PD-1 monoclonal antibody introduced by Jiangsu IlengRui Medicine Co., Ltd. that recently received conditional approval in China for the treatment of relapsed or refractory classical Hodgkin lymphoma.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0095] Sintilimab (IB 1308) is an anti-PD-1 monoclonal antibody developed by Innovent and Eli Lilly for patients with non-small cell lung cancer (NSCLC).

[0096] Tislelizumab (BGB-A317) is a humanized IgG4 anti-PD-1 monoclonal antibody developed by BeiGene for solid tumors and hematologic cancers.

[0097] Dostarlimab (TSR-042, WBP-285) is a humanized monoclonal antibody against PD-1 under investigation by GlaxoSmithKline.

[0098] INCMGA00012 (MGA012) is a humanized IgG4 monoclonal antibody developed by Incyte and MacroGenics.

[0099] AMP-224 is an anti-PD-1 monoclonal antibody by AstraZeneca / Medlmmune and GlaxoSmithKline.

[0100] AMP-514 (MEDI0680) is an anti-PD-1 monoclonal antibody by AstraZeneca.

[0101] A PD-L1 inhibitor is a molecule that binds to and inhibits the biological activity of the PD-L1 protein. Programmed death- ligand 1 (PD-L1) also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that in humans is encoded by the CD274 gene. Examples are anti-PD-Ll antibodies and fragments thereof, such as those described below.

[0102] Atezolizumab (Tecentriq) is a humanized anti-PD-Ll IgGl antibody developed by Roche Genentech. It has been approved by the FDA for urothelial carcinoma and non-small cell lung cancer.

[0103] Avelumab (Bavencio) is a human anti-PD-Ll IgGl antibody developed by Merck Serono and Pfizer. Avelumab has been approved by the FDA for the treatment of metastatic merkel-cell carcinoma.

[0104] Durvalumab (Imfinzi) is a human anti-PD-Ll IgGl antibody developed by AstraZeneca. Durvalumab has been approved by the FDA for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer after chemoradiation.

[0105] KN035 is an anti-PD-Ll antibody with subcutaneous formulation currently under clinical evaluations in the US, China, and lapan.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0106] CK-301 is an anti-PD-Ll antibody being developed by Checkpoint Therapeutics.

[0107] Some small peptide and small molecule inhibitors are also being developed. Examples are shown below.

[0108] AUNP12 is a 29-mer peptide as the first peptic PD-1 / PD-L1 inhibitor developed by Aurigene and Laboratoires Pierre Fabre that is being evaluated in clinical trial, following promising in vitro results.

[0109] CA-170, discovered by Aurigene / Curis as the PD-L1 and VISTA antagonist, was indicted as a potent small molecule inhibitor in vitro. The compound is under phase I clinical trial over mesothelioma patients.

[0110] BMS-986189 is a macrocyclic peptide discovered by Bristol-Myers Squibb of which the pharmacokinetics, safety and tolerability is currently being studied on healthy subjects.

[0111] In some embodiments, the anti-PD-1 inhibitor is pembrolizumab. In some embodiments, the pembrolizumab is administered at 200 mg once every three weeks, or 400 mg once every six weeks. In some embodiments, the cancer is characterized with PD-L1 expression in the tumor. In some the additional therapeutic agent is an RAS inhibitors, a tyrosine kinase inhibitor (TKI), a VEGF / VEGFR inhibitor, a MET inhibitor, a PARP inhibitor, a CDK4 / 6 inhibitor, a bispecific antibody, a chemotherapy, or a radiotherapy.

[0112] In some embodiments, an EGFR-XPAT can be used in combination with a RAS inhibitor. Accordingly, in one embodiment, provided is a method for treating cancer (or enhancing immune cell infiltration into a solid tumor, or improving the efficacy of a checkpoint inhibitor therapy) in a patient in need thereof, wherein the method entails administering to the patient a polypeptide of the instant disclosure (e.g., EGFR-XPAT) and an RAS inhibitor. Another embodiment provides a method for treating cancer in a patient in need thereof, wherein the method entails administering to the patient a polypeptide of the instant disclosure (e.g., EGFR-XPAT) and the patient is further treated with an RAS inhibitor. Another embodiment provides a method for treating cancer in a patient in need thereof, wherein the method entails administering to the patient an RAS inhibitor wherein the patient is further treated with a polypeptide of the instant disclosure (e.g., EGFR-XPAT).Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0113] In some embodiments, the patient being treated has a cancer characterized with a RAS mutation. Examples of RAS mutations include, without limitation, a mutation at codon G12, G13 orQ61 (e.g., G12C).

[0114] Non-limiting examples of RAS inhibitors include AMG510, MRTX849, JNJ-74699157, LY3499446, GDC-6036. BI-1701963, RMC-4630, TNO155, BBP-398, Tipifarnib, LXH-254, Belvarafenib, LXH-254, and BGB-283, as well as those described in, e.g., Conroy et al., Cancer Drug Resist. 2021 Apr 8;4(3):543— 558.

[0115] In some embodiments, the tyrosine kinase inhibitor is lapatinib, erlotinib, gefitinib, imatinib, dasatinib, nilotinib, sunitinib, axitinib, crizotinib, bosutinib, regorafenib, or cabozantinib. In some embodiments, the VEGF / VEGFR inhibitor is bevacizumab, ranibizumab, or aflibercept. In some embodiments, the MET inhibitor is capmatinib, tepotinib, savolitinib, gumarontinib, or vebreltinib. In some embodiments, the PARP inhibitor is olaparib, niraparib, rucaparib, or talazoparib. In some embodiments, the CDK4 / 6 inhibitor is palbociclib, ribociclib, or abemaciclib.

[0116] In some embodiments, the EGFR-XPAT is administered at a dose of at least 1 pg / kg once every week to once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 weeks. In some embodiments, each dose includes at least 2 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes at least 3 pg / kg, 4 pg / kg, 5 pg / kg, 10 pg / kg, 15 pg / kg, 20 pg / kg, 30 pg / kg, 40 pg / kg, 42 pg / kg, 50 pg / kg, 60 pg / kg, 70 pg / kg, 80 pg / kg, 90 pg / kg, 100 pg / kg, 120 pg / kg, 150 pg / kg, 180 pg / kg, 200 pg / kg, 250 pg / kg, 300 pg / kg, 350 pg / kg, 400 pg / kg, 450 pg / kg, 500 pg / kg, 550 pg / kg, 600 pg / kg, 650 pg / kg, 700 pg / kg, 750 pg / kg, 800 pg / kg, 850 pg / kg, or 900 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes no more than 3000 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes no more than 2500 pg / kg, 2000 pg / kg, 1500 pg / kg, 1350 pg / kg, 1300 pg / kg, 1250 pg / kg, 1200 pg / kg, 1150 pg / kg. 1100 pg / kg, 1050 pg / kg, 1000 pg / kg, 950 pg / kg, 900 pg / kg, 850 pg / kg, 800 pg / kg, 750 pg / kg, 700 pg / kg, 650 pg / kg, 600 pg / kg, 550 pg / kg, 500 pg / kg, 450 pg / kg, 400 pg / kg, 250 pg / kg, 200 pg / kg, 150 pg / kg, 120 pg / kg, 100 pg / kg, 90 pg / kg, 80 pg / kg, or 50 pg / kg of the EGFR-XPAT.

[0117] In some embodiments, each dose includes from 1 pg / kg to 1500 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes from 2 pg / kg to 1200 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes from 3 pg / kg to 800 pg / kg of the EGFR-Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO XPAT. In some embodiments, each dose includes from 10 jag / kg to 800 gg / kg of the EGFR-XPAT. In some embodiments, each dose includes from 30 gg / kg to 500 gg / kg of the EGFR-XPAT. In some embodiments, each dose includes from 40 gg / kg to 250 gg / kg of the EGFR-XPAT.

[0118] In some embodiments, the EGFR-XPAT is administered at a dose of at least 0.07 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 120 mg, 125 mg, 150 mg, 175 mg, 180 mg, or 200 mg once every week to once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 weeks. In some embodiments, each dose includes no more than 105 mg of the EGFR-XPAT. In some embodiments, each dose includes no more than 250 mg, 200 mg, 180 mg, 150 mg, 120 mg, 100 mg, 95 mg, 90 mg, 85 mg, 80 mg, 75 mg, 70 mg, 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, 11 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, or 5 mg of the EGFR-XPAT.

[0119] In some embodiments, each dose includes from 0.07 mg to 100 mg of the EGFR-XPAT. In some embodiments, each dose includes from 0.2 mg to 200 mg of the EGFR-XPAT. In some embodiments, each dose includes from 0.1 mg to 80 mg of the EGFR-XPAT. In some embodiments, each dose includes from 0.2 mg to 60 mg of the EGFR-XPAT. In some embodiments, each dose includes from 0.5 mg to 55 mg of the EGFR-XPAT. In some embodiments, each dose includes from 0.7 mg to 50 mg of the EGFR-XPAT. In some embodiments, each dose includes from 1 mg to 45 mg of the EGFR-XPAT. In some embodiments, each dose includes from 2 mg to 40 mg of the EGFR-XPAT. In some embodiments, each dose includes from 3 mg to 10 mg of the EGFR-XPAT. In some embodiments, each dose includes from 5 mg to 15 mg of the EGFR-XPAT. In some embodiments, each dose includes from 10 mg to 30 mg of the EGFR-XPAT. In some embodiments, each dose includes from 30 mg to 50 mg of the EGFR-XPAT. In some embodiments, each dose includes from 30 mg to 70 mg of the EGFR-XPAT.

[0120] In some embodiments, the EGFR-XPAT is administered once every week. In some embodiments, the EGFR-XPAT is administered once every two weeks. In some embodiments, the EGFR-XPAT is administered once every three weeks. In some embodiments, the EGFR-XPAT is administered once every four weeks. In some embodiments, the EGFR-XPAT is administered once every five weeks. In someVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO embodiments, the EGFR-XPAT is administered once every six weeks. In some embodiments, the EGFR-XPAT is administered once every seven weeks. In some embodiments, the EGFR-XPAT is administered once every eight weeks. In some embodiments, the EGFR-XPAT is administered once every nine weeks. In some embodiments, the EGFR-XPAT is administered once every ten weeks.

[0121] In some embodiments, the EGFR-XPAT is administered to a patient once every week and pembrolizumab is administered to the patient once every three weeks. In some embodiments, the EGFR-XPAT is administered to a patient once two weeks and pembrolizumab is administered to the patient once every six weeks. In some embodiments, the EGFR-XPAT is administered to the patient once every three weeks and pembrolizumab is administered to the patient once every three weeks. In some embodiments, the EGFR-XPAT is administered to the patient once every six weeks and pembrolizumab is administered to the patient once every six weeks.

[0122] In a combination therapy, in some embodiments, the EGFR-XPAT is administered at a dose of 30 pg / kg to 1000 pg / kg once every week to once every 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks. In some embodiments, each dose includes at least 30 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes at least 35 pg / kg, 40 pg / kg, 45 pg / kg, 50 pg / kg, 55 pg / kg, 60 pg / kg, 65 pg / kg, 70 pg / kg, 75 pg / kg, 80 pg / kg, 85 pg / kg, 90 pg / kg, 95 pg / kg, 100 pg / kg, 110 pg / kg, 120 pg / kg, 130 pg / kg, 140 pg / kg, 150 pg / kg, 200 pg / kg, 250 pg / kg, 300 pg / kg, 350 pg / kg, 400 pg / kg, 450 pg / kg, 500 pg / kg, 550 pg / kg, 600 pg / kg, 650 pg / kg, 700 pg / kg, 750 pg / kg, 800 pg / kg or 900 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes no more than 1000 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes no more than 900 pg / kg, 850 pg / kg, 800 pg / kg, 750 pg / kg, 700 pg / kg, 650 pg / kg, 600 pg / kg, 550 pg / kg, 500 pg / kg, 450 pg / kg, 400 pg / kg, 350 pg / kg, 300 pg / kg, 250 pg / kg, 200 pg / kg, 150 pg / kg, 140 pg / kg, 130 pg / kg, 120 pg / kg, 110 pg / kg, 100 pg / kg, 90 pg / kg, 85 pg / kg, 80 pg / kg, 75 pg / kg, 70 pg / kg, 65 pg / kg, 60 pg / kg, 55 pg / kg, 50 pg / kg, or 45 pg / kg of the EGFR-XPAT.

[0123] In some embodiments, each dose includes from 30 pg / kg to 1000 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes from 40 pg / kg to 500 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes from 45 pg / kg to 200 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes from 45 pg / kg to 120 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes from 50 pg / kg to 100 pg / kg of the EGFR-Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO XPAT. In some embodiments, each dose includes from 50 jag / kg to 70 pg / kg of the EGFR-XPAT. In some embodiments, each dose includes from 55 pg / kg to 65 pg / kg of the EGFR-XPAT.

[0124] In some embodiments, prior to administration of each dose of EGFR-XPAT to the patient, wherein each dose (also referred to herein as a target dose) is a same amount, the patient receives a first step-up dose less than the target dose before a first administration of the target dose. In some embodiments, the patient receives a second step-up dose after the first step-up dose and before the first administration of the target dose, wherein the second step-up dose is more than the first step-up dose and less than the target dose. In some embodiments, the patient receives a third step-up dose after the second step-up dose and before the first administration of the target dose, wherein the third step-up dose is more than the second step-up dose and less than the target dose.

[0125] In some embodiments, the first step-up dose is administered about one, two, three, four, five, or six weeks before the administration of the first target dose to the patient. In some embodiments, the second step-up dose is administered about one, two, three, four, five, or six weeks before the administration of the first target dose to the patient. In some embodiments, the third step-up dose is administered about one, two, three, four, five, or six weeks before the administration of the first target dose to the patient.

[0126] In some embodiments, a patient is administered a single step-up dose one week prior to the first administration of the target dose. In some embodiments, a patient is administered two step-up doses: a first step-up dose two weeks before the first administration of the target dose and the second step-up dose one week before the first administration of the target dose. In some embodiments, the patient is administered three step-up doses: a first step-up dose three weeks before the first administration of the target dose, a second step-up dose two weeks before the first administration of the target dose, and a third step-up dose one week before the first administration of the target dose.

[0127] In some embodiments, the first administration of the target dose may occur less than two weeks (for a Q2W dosing regiment, or less than three weeks for a Q3W dosing regimen) before the second administration of the target dose, regardless of the dosing regime thereafter. Such a design allows the subsequent target doses (i.e., the second target and later ones) to be administered on the same schedule as if no step-up doses were administered. ForVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO example, as illustrated in FIG. 16B, three step-up doses (once per week) are administered to the patient for a Q2W dosing regimen, such that the first target dose is administered on day 22 (D22), the beginning of the 4thweek. Without such step-up doses, the subsequent target dose would be at the beginning of the 5thweek. In order to keep this schedule, the second target dose actually is administered just one week after the first target dose, such that it is still administered at the beginning of the 5thweek. All subsequent doses, however, are two weeks apart from one another.

[0128] In some embodiments, the patient has received an initial therapy prior to administration of the EGFR-XPAT (i.e., a premedication). In some embodiment the premedication is administered to the patient before the first dose of the EGFR-XPAT. In some embodiments, the premedication is administered to the patient before each dose of the EGFR-XPAT. In some embodiments, the initial therapy is an antihistamine, an antipyretic, a steroid, an IL-6 receptor antagonist, and any combination thereof. In some embodiments, the antihistamine is diphenhydramine, cetirizine, promethazine, or dexchlorpheniramine. In some embodiments, the antipyretic is acetaminophen (aka paracetamol). In some embodiments, the steroid is a corticosteroid. In some embodiments, the corticosteroid is dexamethasone or prednisolone. In some embodiments, the premedication is administered to the patient between 30 minutes and two hours prior to administration of the EGFR-XPAT. In some embodiments, the premedication is administered to the patient between 30 minutes and 60 minutes prior to administration of the EGFR-XPAT. In some embodiments, the premedication is administered to the patient one, two, three, four, or five hours prior to administration of the EGFR-XPAT.Cancer Treatments and Treatment Monitoring

[0129] The compositions, methods and treatment regimens are useful for treating various cancer types, in particular those characterized with EGFR expression. In some embodiments, a patient treated by the methods described herein has a solid tumor characterized by expression of EGFR. In some embodiments, the solid tumor overexpresses EGFR. In some embodiments, the solid tumor is characterized by EGFR gene amplification.

[0130] EGFR is frequently expressed in lunger cancer. Lung cancer is the second-most diagnosed cancer and leading cause of cancer deaths worldwide. As with many other cancers, histological subtyping and molecular profiling has revealed significant heterogeneity in lungVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO cancer. From a histological perspective, approximately 85% of lung cancers are NSCLC and 15% are small -cell lung cancer. NSCLC patients can be further segmented into 3 major histological subtypes, which include adenocarcinoma, squamous cell, and large -cell lung cancer. Molecular profiling of NSCLC tumors in recent decades has further elucidated disease biology and uncovered several driver mutations that have been successfully targeted with therapeutic agents. Unfortunately, despite advances, lung cancer patients still have an overall poor prognosis with an estimated 5-year survival rate from diagnosis of only 10% to 20% in most countries.

[0131] Alterations in EGFR are the most common targetable drivers in NSCLC, with prevalence ranging from approximately 15% in the United States / Europe to upwards of 60% in certain Asian regions. The most common types of EGFR-activating alterations in NSCLC are exon 19 deletions, L858R mutation, T790M mutation, and exon 20 insertions. EGFR mutations are often accompanied by increases in EGFR copy number. Tyrosine kinase inhibitors (TKIs), such as the third -generation TKI osimertinib, have demonstrated impressive anti -tumor activity in patients with exon 19 and L858R alterations and are now standard of care in front-line locally advanced / metastatic and adjuvant disease. Mobocertinib, a novel TKI, and amivantamab, a bispecific mAb targeting EGFR andmesenchymal-epithelial transition (MET), were also recently granted accelerated approval specifically for the treatment of EGFR exon 20 insertions.

[0132] Despite EGFR reportedly being overexpressed in 50% to 90% of NSCLC, efforts to target EGFR with mAbs in EGFR wild-type NSCLC have achieved limited success. In the FLEX Phase 3 trial, cetuximab in combination with platinum -based chemotherapy modestly prolonged median OS (hazard ratio [HR]: 0.87) and increased objective response rate (ORR; 36% vs 29%) compared with chemotherapy alone. Correlative studies based on EGFR protein expression indicated that clinical benefit was enriched in patients whose tumors exhibited high EGFR (HR: 0.73) compared with those with low EGFR (HR: 0.99).Necitumumab, a second -generation EGFR antibody that induces fewer hypersensitivity reactions than cetuximab, has also been evaluated. In the SQUIRE Phase 3 trial, necitumumab in combination with gemcitabine and cisplatin improved OS (HR: 0.84) compared with gemcitabine and cisplatin alone in front-line squamous NSCLC and was approved by regulatory bodies. EGFR targeting agents in EGFR wild-type NSCLC are currently not a mainstay therapy.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0133] For NSCLC patients who do not harbor actionable driver mutations, immunotherapy with or without chemotherapy is the current standard of care for first -line treatment of locally advanced or metastatic disease. Single -agent anti-programmed cell death (ligand) 1 [PD-(L)1] antibodies, such as pembrolizumab and atezolizumab, are typically prescribed in patients with programmed cell death ligand 1 (PD-L1) expression %5()%. For patients with PD-L1 expression <50% and patients with PD-L1 ^50% and rapidly progressive or extensive disease, immunotherapy (anti -PD-[L]1 ± anti-cytotoxic T-lymphocyte -associated protein 4 [CTLA-4] or ± bevacizumab) in combination with a platinum -doublet chemotherapy is the preferential therapeutic option. Notably, histological subtype influences the choice of chemotherapy partner.

[0134] Unfortunately, patients with or without EGFR aberrations almost inevitably relapse from their disease and are subsequently treated with chemotherapy. Patients receiving a third -generation TKI typically receive a platinum -based therapy, while patients refractory to immunotherapy and platinum -based chemotherapy receive docetaxel ± ramucirumab. There is a significant unmet need to develop new therapeutic options for patients with and without EGFR aberrations.

[0135] EGFR is also frequently expressed in colorectal cancer. Colorectal cancer represents the third-most-common and second -most -deadly cancer, with approximately 1.9 million new cases and 935,000 deaths globally in 2020. The overall prognosis of patients with metastatic CRC is poor, with a 5-year survival rate of less than 20%. Molecular profiling of CRC tumors has yielded new insights into disease biology and expedited the development of new therapeutic options. Mutations in KRAS, NRAS, or BRAF are found in approximately, 44%, 4%, and 9% of CRC patients, respectively, and influence treatment decisions and choice of targeted biological agent(s). Furthermore, EGFR is estimated to be overexpressed in 60% to 80% of CRC tumors and is associated with a poor prognosis. For these reasons, EGFR and downstream signaling pathways have been extensively targeted.

[0136] The initial treatment for unresectable metastatic CRC patients includes a systemic chemotherapy backbone (fluoropyrimidines, oxaliplatin, and irinotecan) paired with a targeted biological agent (anti-vascular endothelial growth factor [VEGF] or anti -EGFR antibody). Typical regimens include 5-fluorouracil (5-FU) / leucovorin (LV) / oxaliplatin (FOLFOX), capecitabine / LV / oxaliplatin (CAPOX), or 5-FU / LV / irinotecan (FOLFIRI). Patients who received irinotecan regimens in first -line treatment (e.g., FOEFIRl) are usuallyVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO switched to oxaliplatin-containing regimens (e.g., FOLFOX or CAPOX), whereas patients treated with FOLFOX or CAPOX in first-line treatment are treated with irinotecan monotherapy or FOLFIRI.

[0137] Anti-EGFR therapies (.e.g., cetuximab or panitumumab) can be given in patients receiving first- line treatment with chemotherapy who have RAS / BRAF wild-type mutations or in later lines of therapy as a single agent or in combination with irinotecan. Anti-VEGF agents (e.g., bevacizumab) can be given in the first- or second -line setting with chemotherapy regardless of mutation status. For patients with BRAF mutations, targeted agents consisting of cetuximab plus encorafinib (BRAF inhibitor) are given as second -line treatment. In the third -line or later setting, the treatment options are usually single -agent therapies, such as regorafenib or TAS-102, and survival benefit is typically less than 1 year. In Phase 3 trials, CRC patients treated with regorafenib or TAS-102 displayed an ORR of 1% or 1.6%, a median PFS of 2 months, and a median OS of 6.4 or 7.1 months, respectively. It is noteworthy that efficacy in the KRAS -mutant population was worse than in the overall population. Recently, TAS-102 plus bevacizumab was also approved by the Food and Drug Administration (FDA) and European Medicines Agency for the third -line or later setting. In the SUNLIGHT Phase 3 trial, patients treated with TAS-102 plus bevacizumab had a median OS of 10.8 months vs 7.5 months in patients treated with TAS-102 alone.

[0138] With the exception of immune checkpoint blockade in microsatellite instability-high (MSLII) or deficient mismatch repair (dMMR) tumors, immunotherapy approaches for CRC treatment have seen limited success. However, combination studies with immunotherapy continue to be evaluated. The demonstrated lack of effective therapies in patients with previously treated CRC and overall poor disease prognosis highlights a high unmet medical need.

[0139] Head and neck cancers are the sixth-most-common cancer worldwide, with an estimated 890,000 new cases and 450,000 deaths in 2018. Head and neck cancers are a heterogeneous group that originate from mucosal epithelium and affect the oral cavity, oropharynx, larynx, hypopharynx, and nasopharynx. Squamous cell carcinoma is the most predominant histological subtype and accounts for over 90% of all malignant disease in the head and neck region. Risk factors include tobacco use, alcohol consumption, and human papillomavirus (HPV) infection. HPV-associated HNSCCs tend to have better overallVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO prognosis, are mostly restricted to the oropharynx in origin, and exhibit lower EGFR expression than do those in HPV-negative patients.

[0140] HNSCCs exhibit significant EGFR upregulation and high frequency of EGFR copy number gain, and, unlike NSCLC, a low rate of genetic EGFR mutation (e.g., insertions, deletions, point mutations). EGFR is reportedly overexpressed in >90% of head and neck tumors, and overexpression is generally associated with shorter OS. Due to the high levels of EGFR expression, anti-EGFR therapy, including TKIs and mAbs, has been extensively investigated in HNSCC patients with mixed results. Cetuximab is the only marketed anti -EGFR therapy in HNSCC and was initially approved for the treatment of local or regionally advanced disease in combination with radiation therapy or as a monotherapy for the treatment of recurrent or metastatic disease progressing after platinum -based chemotherapy. Despite the high expression of EGFR, cetuximab provides nominal improvement in HNSCC, with response rates of 13% as a monotherapy following progression after platinum-based chemotherapy. Subsequently, cetuximab was also approved for recunent locoregional or metastatic disease in combination with platinum -based chemotherapy with fluorouracil.

[0141] The immune checkpoint inhibitor pembrolizumab was recently approved by the FDA as monotherapy (for patients with a PD-L1 combined positive score of 1 %) and in combination with platinum and fluorouracil for the first -line treatment of patients with metastatic or unresectable recunent HNSCC. In the KEYNOTE-048 study, pembrolizumab plus platinum and fluorouracil increased median OS by approximately 2.3 months (13.0 mo vs 10.7 mo; HR: 0.77). Pembrolizumab and nivolumab are also approved by the FDA for treatment of cisplatin -refractory recunent or metastatic HNSCC.

[0142] Unfortunately, HNSCC patients treated with immunotherapy and platinum-based therapy almost inevitably become refractory, and there is currently no clear second -line option for metastatic patients. Therapy options include chemotherapy, cetuximab monotherapy, or cetuximab plus taxane-based regimens. There is a high unmet need to develop novel therapies for the treatment of HNSCC.

[0143] The generation of large genomic datasets across tumor types has enabled a thorough characterization of EGFR genetic mutation and copy number variation. In a recent analysis using The Cancer Genome Atlas dataset, it was found that the overall EGFR amplificationVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO frequency across all analyzed tumor types was 4.8% (549 of 11,410 samples). Glioblastoma (43.9%), esophageal cancer (14.1%), head and neck cancer (10.9%), and lung squamous adenocarcinoma (7.6%) were the most common solid tumors with EGFR amplification. In a separate study, investigators evaluated circulating tumor DNA (ctDNA) samples across diverse cancers and found that EGFR was amplified in 8.5% of total patients (2424 of 28,584 samples). EGFR amplification was most commonly found in CRC (16.3%), NSCLC (9%), and genitourinary cancers (8.1%). Investigators also discovered EGFR amplification emerging in longitudinal ctDNA samples following anti-cancer therapy, indicative of potential acquired resistance mechanisms. Importantly, EGFR amplification offers an opportunity to enrich for patients with elevated EGFR expression and test XPAT01 in a spectrum of solid tumors. Any of such cancers can be suitably treated with the instant technology.

[0144] EGFR expression and / or EGFR gene amplification can be measured in a tumor biopsy. Tests can be performed on biopsy samples obtained by either fine-needle aspiration, core needle biopsy, vacuum-assisted breast biopsy, or surgical excision. In some embodiments, EGFR gene amplification can be determined using in situ hybridization.

[0145] Immunohistochemistry (IHC) can be used to measure the amount of EGFR protein present in the sample, with fluorescence in situ hybridization (FISH) being used on samples that are equivocal in IHC. A sample IHC scoring system is described in Table A.Table A. EGFR IHC ScoresScore PatternEither:— No staining observed.0— Incomplete membrane staining that is faint or barely perceptible and within<10% of the invasive tumor cellsIncomplete membrane staining that is faint or barely perceptible and within >10% of1 +the invasive tumor cells.2+ Weak to moderate complete membrane staining observed in >10% of tumor cells.Circumferential membrane staining that is complete, intense, and in >10% of tumor3+cells.

[0146] In an example scoring system, in some embodiments, the patient has a EGFR+cancer. In some embodiments, the EGFR+cancer has an immunohistochemistry (IHC) grade 2+. In some embodiments, the EGFR+cancer has an immunohistochemistry (IHC) grade 3+. InVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO some embodiments, the EGFR+cancer is in situ hybridization positive (ISH+). In some embodiments, the EGFR+cancer has one or more EGFR mutations.

[0147] In some embodiments, the patient has had one or more lines of prior treatments. In some embodiments, the patient is resistant to or has relapsed from such prior treatments. In one embodiment, the patient has had at least 1 line of prior treatment and is resistant to or has relapsed from the at least 1 line of prior treatment. In one embodiment, the patient has had at least 2 lines of prior treatment and is resistant to or has relapsed from the at least 2 lines of prior treatment. In one embodiment, the patient has had at least 3 lines of prior treatment and is resistant to or has relapsed from the at least 3 lines of prior treatment. In one embodiment, the patient has had at least 4 lines of prior treatment and is resistant to or has relapsed from the at least 4 lines of prior treatment. In one embodiment, the patient has had at least 5 lines of prior treatment and is resistant to or has relapsed from the at least 5 lines of prior treatment.

[0148] In one embodiment, the patient has had at least 6 lines of prior treatment and is resistant to or has relapsed from the at least 6 lines of prior treatment. In one embodiment, the patient has had at least 7 lines of prior treatment and is resistant to or has relapsed from the at least 7 lines of prior treatment. In one embodiment, the patient has had at least 8 lines of prior treatment and is resistant to or has relapsed from the at least 8 lines of prior treatment. In one embodiment, the patient has had at least 9 lines of prior treatment and is resistant to or has relapsed from the at least 9 lines of prior treatment. In one embodiment, the patient has had at least 10 lines of prior treatment and is resistant to or has relapsed from the at least 10 lines of prior treatment.

[0149] In some embodiments, at least one of the prior treatments is a standard of care (SOC) treatment for the cancer type. In some embodiments, at least one of the prior treatments includes chemotherapy. In some embodiments, at least one of the prior treatments includes radiotherapy. In some embodiments, at least one of the prior treatments includes an anti-EGFR treatment. In some embodiments, the anti-EGFR therapy comprises one or more of an anti-EGFR antibody or small molecule inhibitor, such as gefitinib, erlotinib, afatinib, brigatinib, icotinib, cetuximab, osimertinib, panitumumab, zalutumumab, nimotuzumab, and matuzumab.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0150] In some embodiments, the cancer is breast cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, ovarian cancer, bladder cancer, colorectal cancer (CRC), endometrial cancer, head and neck cancer, lung cancer, prostate cancer, liver cancer, brain cancer, cervical cancer, kidney cancer, pancreatic cancer, skin cancer, or salivary gland cancer. In some embodiments, the brain cancer is glioblastoma. In some embodiments, the CRC is microsatellite stable (MSS). In some embodiments, the CRC patient has wild-type BRAE In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is squamous NSCLC. In some embodiments, the NSCLC has EGFR-activating mutation. In some embodiments, the head and neck cancer is head and neck squamous cell carcinomas (HNSCC). In some embodiments, the skin cancer is cutaneous squamous cell carcinoma (CSCC). In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the kidney cancer is renal cell carcinoma (RCC).

[0151] In some embodiments, the cancer is anaplastic and medullary thyroid cancers, appendiceal cancer, arrhenoblastoma, biliary tract carcinoma, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignancy with malignant ascites, esophageal cancer, Ewing sarcoma, fallopian tube cancer, follicular cancer, gall bladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), GE-junction cancer, genito-urinary tract cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocarcinoma, HR+ and HER2+ breast cancer, Hurthle cell cancer, Inflammatory breast cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroblastoma, neuroendocrine cancer, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary cancer, parathyroid cancer, peritoneal carcinomatosis, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, triple negative breast cancer, urothelial cancer, uterine cancer, uterine serous carcinoma, vaginal cancer, vulvar cancer, or Wilms tumor.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0152] As noted before, even at very high doses, XPAT01 only caused low level CRS. Therefore, no prophylactic use of steroids is needed to prevent or treat such low level CRS. In one embodiment, therefore, prior to the first dose of the EGFR-XPAT, the patient is not administered a prophylactic dose of a steroid. In some embodiments, during the treatment with the EGFR-XPAT, the patient is not also treated with a steroid.

[0153] In some embodiments, even when a steroid premedication is used, it can be used at a relatively low dose. For instance, the steroid can be a corticosteroid, such as dexamethasone. In some embodiments, the dexamethasone is used at no more than 20 mg every 6 hours or longer. In some embodiments, the dexamethasone is used at no more than 10 mg every 6 hours or longer. In some embodiments, the dexamethasone is used at no more than 5 mg every 6 hours or longer.

[0154] In some embodiments, a steroid is used once a CRS sign or biomarker is detected. For instance, a steroid is administered after an CRS episode, or detection of pneumonitis. In some embodiments, a steroid is administered when an increase of TL-6 expression is detected.

[0155] In some embodiments, methods are also provided for monitoring the progress of the cancer treatment, or for predicting the progress. In one embodiment, once a decrease of EGFR level is detected, e.g., in the blood or another body fluid, it can be predicted that the patient is responding to the EGFR-XPAT treatment and tumor shrinkage will follow. In some embodiments, the biomarker is GM-CSF, IFN-y, IL- 1 , IL-2, IL-4, IL-6, IL- 10, MCP-1, or TNF-a.EGFR-XPAT

[0156] An example polypeptide suitable for use in the instant methods is EGFR-XPAT, which contains a TCE (T-cell engager) core having dual specificity to EGFR and CD3, flanked by a N-terminal and a C-temiinal unstructured polypeptide masks (XTENs).

[0157] “Extended recombinant polypeptides” (or XTEN) as used herein, are polypeptides with non-naturally occurring, substantially non-repetitive sequences having a low degree or no secondary or tertiary structure under physiologic conditions. XTEN typically have from at least about 100 to at least about 1000 or more amino acids, the majority or the entirety of which are small hydrophilic amino acids. As used herein, XTEN specifically excludes wholeVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO antibodies or antibody fragments (e.g. single-chain antibodies and Fc fragments). Examples of XTEN can be found in W02017040344, without limitation.

[0158] In one embodiment, the EGFR-XPAT includes (a) a first extended recombinant polypeptide (XTEN) having at least 70% sequence identity to SEQ ID NO:2, (b) a core fragment comprising an anti-EGFR antibody or antigen-binding fragment and an anti-CD3 antibody or antigen-binding fragment, and (c) a second XTEN having at least 70% sequence identity to SEQ ID NO: 12. In some embodiments, (a) is connected to (b) through a first protcasc-clcavablc site, and (b) is connected to (c) through a second protcasc-clcavablc site.

[0159] In some embodiments, the first XTEN has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2. In some embodiments, the first XTEN includes the sequence of SEQ ID NO:2. In some embodiments, the second XTEN has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the second XTEN includes the sequence of SEQ ID NO:12.

[0160] In some embodiments, the anti-EGFR antigen-binding fragment include a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH includes the VH CDRs of SEQ ID NO:6. In some embodiments, the VH includes such VH CDR sequences and have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:6. In some embodiments, the VL includes the VL CDRs of SEQ ID NO:5. In some embodiments, the VL includes such VL CDR sequences and have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:5.

[0161] In some embodiments, the anti-EGFR VH and the anti-EGFR VL are connected via a first peptide linker. In some embodiments, the linker has a sequence of SEQ ID NO:9 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:9.

[0162] In some embodiments, the anti-EGFR antigen-binding fragment has a VL-linker-VH configuration. In some embodiments, the anti-EGFR antigen-binding fragment has a VH-linker-VL configuration. In some embodiments, the anti-EGFR antigen-binding fragment has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ 1D NO:13.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0163] In some embodiments, the anti-CD3 antigen-binding fragment include a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH includes the VH CDRs of SEQ ID NO: 8. In some embodiments, the VH includes such VH CDR sequences and have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the VL includes the VL CDRs of SEQ ID NO:7. In some embodiments, the VL includes such VL CDR sequences and have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:7.

[0164] In some embodiments, the anti-CD3 antigen-binding fragment includes a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:8 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:7.

[0165] In some embodiments, the anti-CD3 VH and the anti-CD3 VL are connected via a second peptide linker. In some embodiments, the linker has a sequence of SEQ ID NO: 9 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:9.

[0166] In some embodiments, the anti-CD3 antigen-binding fragment has a VL-linker-VH configuration. In some embodiments, the anti-CD3 antigen-binding fragment has a VH-linker-VL configuration. In some embodiments, the anti-CD3 antigen-binding fragment has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:14.

[0167] In some embodiments, the anti-EGFR antibody or antigen-binding fragment is connected to the anti-CD3 antibody or antigen-binding fragment through a peptide linker. In some embodiments, the linker has a sequence of SEQ ID NO: 10. Together, these elements form a T-cell engager core. In some embodiments, the T-cell engager core includes the anti-EGFR antibody or antigen-binding fragment, the linker, and the anti-CD3 antibody or antigen-binding fragment, from N-tcrminus to C-tcrminus. In some embodiments, the T-cell engager core includes the anti-CD3 antibody or antigen-binding fragment, the linker, and the anti-EGFR antibody or antigen-binding fragment, from N-terminus to C-terminus.

[0168] In some embodiments, the T-cell engager core has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the T-cell engager core has the sequence of SEQ ID NO: 15.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0169] In some embodiments, the T-cell engager core is connected to the first XTEN via a first protease cleavable site, and optionally a first spacer. In some embodiments, the T-cell engager core is connected to the second XTEN via a second protease cleavable site, and optionally a second spacer. In some embodiments, the first protease cleavable site and the second protease cleavage site is clcavablc by a protease present in a tumor microenvironment. In some embodiments, the protease is selected from MMP-2, MMP-7, MMP-9, MMP-13, MMP-14, urokinase (uPA), and matriptase. In some embodiments, the first protease cleavable site and the second protease cleavage site each independently has the sequence of SEQ ID NO:3 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3. In some embodiments, the first spacer has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:4. In some embodiments, the second spacer has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:11.

[0170] In some embodiments, the EGFR-XPAT includes the elements as disclosed herein and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1. In some embodiments, the EGFR-XPAT is XPAT01 which includes the sequence of SEQ ID NO: 1.Pharmaceutical Compositions

[0171] Disclosed herein includes a pharmaceutical composition comprising a polypeptide, such as the EGFR-XPAT described hereinabove or described anywhere else herein, and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, oral, intravenous, intra-arterial, intraabdominal, intraperitoneal, intravitreal, intrathecal, or intramuscular administration. In some embodiments, the pharmaceutical composition is in a liquid form or frozen. In some embodiments, the pharmaceutical composition is in a device that is implanted into the eye or another body part. In some embodiments, the pharmaceutical composition is in a pre-filled syringe for a single injection. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder to be reconstituted prior to administration.

[0172] In some embodiments, the dose is administered intradermally, subcutaneously, orally, intravenously, intravitreally (or otherwise injected into the eye), intra-arterially, intra-abdominally, intraperitoneally, intrathecally, or intramuscularly. In some embodiments, theVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO dose is administered intravenously. In some embodiments, the dose is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered using a device implanted into the eye or other body part. In some embodiments, the subject is a mouse, rat, monkey, or human.

[0173] The pharmaceutical compositions can be administered for therapy by any suitable route. In addition, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.

[0174] In certain embodiments, the polypeptide of the instant disclosure (e.g., EGFR-XPAT) may be used in combination with (or further in combination with, is already used along with an anti-PD-1 or anti-PD-Ll inhibitor) a second therapeutic agent effective for treating or ameliorating the effects of the cancer. The additional therapeutic agent may be selected from the group consisting of an antibody, an antibody fragment, an antibody conjugate, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof. Particularly referred second or additional therapeutic agents include other EGFR targeting agents, chemotherapy agents, radiotherapeutic agents, as well as agents that target HER3 and other targets that are involved in resistance to treatment of EGFR-driven cancers.

[0175] Examples of therapeutic antibodies that may be used in the present invention include rituximab (Rituxan), Brentuximab Vedotin (Adcetriz), Ado-trastuzumab emtansine (Kadcyla), Cetuximab (Erbitux), bevacizumab (Avastin), Ibritumomab (Zevalin), vedolizumab (Entyvio), Ipilimumab (Yervoy), Nivolumab (Opdivo), pembrolizumab (Keytruda), Alemtuzamab atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), B-701, Ofatumumab, Obinutuzumab (Gazyva) Panitumumab, plozalizumab, BI-754091, OREG-103, COM-701, BI-754111, and combinations thereof.

[0176] According to some embodiments, the antibody, fragment thereof, or conjugate thereof is selected from the group consisting of rituximab (Rituxan), Brentuximab Vedotin (Adcetriz), Ado-trastuzumab emtansine (Kadcyla), Ipilimumab (Yervoy), Nivolumab (Opdivo), pembrolizumab (Keytruda), Alemtuzamab atezolizumab (Tecentriq), durvalumab (Imfinzi), Ofatumumab, Obinutuzumab (Gazyva) Panitumumab, and combinations thereof.

[0177] In other embodiments, the additional agent may be a DNA damaging agent, antimetabolite, anti-microtubule agent, antibiotic agent, etc. DNA damaging agents includeVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO alkylating agents, platinum-based agents, intercalating agents, and inhibitors of DNA replication. Non-limiting examples of DNA alkylating agents include cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Non-limiting examples of platinum-based agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Non-limiting examples of intercalating agents include doxorubicin, daunorubicin, idarubicin, mitoxantrone, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Non-limiting examples of inhibitors of DNA replication include irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Antimetabolites include folate antagonists such as methotrexate and premetrexed, purine antagonists such as 6-mercaptopurine, dacarbazine, and fludarabine, and pyrimidine antagonists such as 5-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, decitabine, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Anti-microtubule agents include without limitation vinca alkaloids, paclitaxel (Taxol®), docetaxel (Taxotere®), and ixabepilone (Ixempra®). Antibiotic agents include without limitation actinomycin, anthracyclincs, valrubicin, cpirubicin, bleomycin, plicamycin, mitomycin, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.

[0178] Exemplary cytotoxic agents are known to those of skill in the art, and may, for example, be selected from the group consisting of cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, doxorubicin, daunorubicin, idarubicin, mitoxantrone, methotrexate, pemetrexed, 6-mercaptopurine, dacarbazine, fludarabine, 5-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, decitabine, vinca alkaloids, paclitaxel (Taxol), docetaxel (Taxotere), ixabepilone (Ixempra), actinomycin, anthracy clines, valrubicin, epirubicin, bleomycin, plicamycin, mitomycin, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.

[0179] Cytotoxic agents according to the present invention also include an inhibitor of the PI3K / Akt pathway. Non-limiting examples of an inhibitor of the PI3K / Akt pathway includeVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO A-674563 (CAS #552325-73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, Calif.), AS-041164 (5-benzo[l, 3 ]dioxol-5-ylmethylene-thiazolidine-2, 4-dione), AS-604850 (5-(2,2-Difluoro-benzo[l, 3 ]dioxol-5-ylmethylene)-thiazolidine-2, 4-dione), AS-605240 (5-quinoxilin-6-methylene-l,3-thiazolidine-2, 4-dione), AT7867 (CAS #857531-00-1), benzimidazole scries, Gcncntcch (Roche Holdings Inc., South San Francisco, Calif.), BML-257 (CAS #32387-96-5), BVD-723, CAL- 120 (Gilead Sciences, Foster City, Calif.), CAL- 129 (Gilead Sciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (Gilead Sciences), CAS #612847-09-3, CAS #681281-88-9, CAS #75747-14-7, CAS #925681-41-0, CAS #98510-80-6, CCT128930 (CAS #885499-61-6), CH5132799 (CAS #1007207-67-1), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK), FPA 124 (CAS #902779-59-3), GS-1101 (CAL-101) (Gilead Sciences), GSK 690693 (CAS #937174-76-0), H-89 (CAS #127243-85-0), Honokiol, IC87114 (Gilead Science), IPL145 (Intellikine Inc.), KAR-4139 (Karns Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1 (Kams Therapeutics), KT 5720 (CAS #108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS #1032350-13-2), ML-9 (CAS #105637-50-1), Naltrindole Hydrochloride, OXY-1 HA (NormOxys Inc., Brighton, Mass.), perifosine, PHT-427 (CAS #1191951-57-1), PI3 kinase delta inhibitor, Merck KGaA (Merck & Co., Whitehouse Station, N.J.), PI3 kinase delta inhibitors, Gcncntcch (Roche Holdings Inc.), PI3 kinase delta inhibitors, Incozcn (Incozcn Therapeutics, Pvt. Ltd., Hydrabad, India), PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (Roche Holdings Inc.), PI3-alpha / delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd., South San Francisco, Calif.), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-delta inhibitors, Intellikine (Intellikine Inc., La Jolla, Calif.), PI3-delta inhibitors, Pathway Therapeutics- 1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta / gamma inhibitors, Cellzome (Cellzome AG), PI3-delta / gamma inhibitors, Cellzome (Cellzome AG), PI3-delta / gamma inhibitors, Intellikine (Intellikine Inc.), PI3-delta / gamma inhibitors, Intellikine (Intellikine Inc.), PI3-delta / gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3 -delta / gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitor Evotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3 -gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3K delta / gamma inhibitors, Intellikine-1 (Intellikine Inc.), PI3K delta / gamma inhibitors, Intellikine-1 (Intellikine Inc.), pictilisib (Roche Holdings Inc.), PIK-90 (CAS #677338-12-4), SC-103980 (Pfizer, New York, N.Y.),Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO SF-1126 (Semafore Pharmaceuticals, Indianapolis, Ind.), SH-5, SH-6, Tetrahydro Curcumin, TG100-115 (Targegen Inc., San Diego, Calif.), Triciribine, X-339 (Xcovery, West Palm Beach, Fla.), XL-499 (Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof, and combinations thereof.

[0180] The additional agent in the combination therapy may be a poison or venom of plant or animal origin. An example is diphtheria toxin or portions thereof. In other examples, the additional agent may be a “radionuclide” i.e., a radioactive substance administered to the patient, e.g., intravenously or orally, after which it penetrates via the patient's normal metabolism into the target organ or tissue, where it delivers local radiation for a short time. Examples of radionuclides include, but are not limited to, 1-125, At-211, Lu-177, Cu-67, 1-131, Sm-153, Re-186, P-32, Re-188, In-114m, and Y-90.

[0181] The term “immunomodulator” means a substance that alters the immune response by augmenting or reducing the ability of the immune system to produce antibodies or sensitized cells that recognize and react with the antigen that initiated their production.Immunomodulators may be recombinant, synthetic, or natural preparations and include cytokines, corticosteroids, cytotoxic agents, thymosin, and immunoglobulins. Some immunomodulators are naturally present in the body, and certain of these are available in pharmacologic preparations. Examples of immunomodulators include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), LAG-3, IMP-321, ICAR-014, ASLAN-002 (BMS-777607), interferons, imiquimod and cellular membrane fractions from bacteria, IL-2, IL-7, IL- 12, CCL3, CCL26, CXCL7, synthetic cytosine phosphate-guanosine (CpG), immune-checkpoint inhibitors, and combinations thereof. Targeted cytokine therapy, particularly targeting to lymphotoxin and LiGHT may also be useful in combination with the compositions of the present invention.

[0182] In some combination treatments, the additional agent may be a “radiosensitizing agent” that makes tumor cells more sensitive to radiation therapy. Examples of radiosensitizing agents include misonidazole, metronidazole, tirapazamine, and trans sodium crocetinate, and combination thereof.

[0183] In still other embodiments, the additional agent is an “anti-angiogenesis” agent that reduces or inhibits the growth of new blood vessels, such as, e.g., an inhibitor of vascular endothelial growth factor (VEGF) and an inhibitor of endothelial cell migration. Anti-Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO angiogenesis agents include without limitation 2-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-a, IL- 12, itraconazole, linomide, platelet factor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.EXAMPLESExample 1: Non-Clinical Evaluation of XPAT01

[0184] Multiple studies were conducted to characterize the pharmacology, pharmacokinetics (PK), pharmacodynamics, and toxicity of the investigational prodrug and clinical candidate, XPAT01.

[0185] XPAT01 (SEQ ID NO:1, see Table 1) is a human epidermal growth factor receptor (EGFR)-targeted, protease-activated T cell engager (TCE), or XTEN polypeptide-fused protease- activated TCE (XPAT) designed to exploit the dysregulated protease activity in tumors, while sparing healthy tissues where there is minimal protease activity, thus broadening the safety margin and therapeutic index. XPAT01 consists of a core of two tandem single-chain variable fragments (scFvs) targeting cluster of differentiation (CD) 3 and EGFR. The core is flanked by two unstructured protein polymer masks (XTENs, SEQ ID NO:2 and 12) designed to extend half-life (ti / 2) and sterically block target engagement of either EGFR or CD3. Protease cleavage sites (SEQ ID NOG) at the base of each XTEN in XPAT01 enable preferential proteolytic activation of XPAT01 in the tumor microenvironment, unleashing a small, highly potent unmasked TCE core, referred to as XPAT01-TCE (SEQ ID NO:15), able to redirect cytotoxic T cells to kill target-expressing tumor cells. Singly cleaved species retain an N- or C-terminal mask (hence XPATOl(lx-N) or XPATOl(lx-C)) and exhibit activity intermediate to XPAT01-TCE and fully masked XPAT01. In healthy tissues, where protease activity is tightly regulated, XPAT01 remains predominantly inactive as a fully masked protein.Table 1. XPAT01 SequencesName Sequence SEQ ID NO:XPAT01 ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSES 1ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSASHTPAG LTGPGTSESATPESDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQ QKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QHFDHLPLAFGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQV QLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHI YYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTG AFD IWGQGTLVTVSSGGGGSELWTQEPSLTVSPGGTVTLTCRSSNGAVTS SNYANWQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQP EDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGT SESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGL EWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVY YCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAG LTGPATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESAGEPEAN-terminal ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSES 2 XTEN ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPFirst protease EAGRSASHTPAGLTGP 3 cleavable siteFirst spacer GTSESATPES 4 Anti-EGFR DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDA 5VL SNLETGVPSRFSGSGSGTDFTFTISSLQPED IATYYCQHFDHLPLAFGQGT KVEIKFirst scFv SESATPESGPGTSPGATPESGPGTSESATP 9 linkerAnti-EGFR QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIG 6 VH HIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRV TGAFDIWGQGTLVTVSSInter-scFv GGGGS 10 linkerAnti-CD3 VL ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIG 7GTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG GTKLTVLSecond scFv SESATPESGPGTSPGATPESGPGTSESATP 9linkerVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Anti-CD3 VH EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRI 8RTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHE NFGNSYVSWFAHWGQGTLVTVSSSecond spacer GTATPESGPG 11 Second EAGRSASHTPAGLTGP 3 proteasecleavable siteC-terminal ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG 12 XTEN SETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGTSPSATPESGPGSEPATSGSET PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS EPATSGSETPGTSESAGEPEAAnti-EGFR DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDA 13 scFv SNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGTKVE IKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVKPSE TLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHI YYSGNTNYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVS SAnti-CD3 ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIG 14 scFv GTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG GTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQLVESGGGIVQP GGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADS VKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWG QGTLVTVSS XPAT01 TCE DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDA 15SNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGT KVE IKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVKPSE TLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHI YYSGNTNYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVS SGGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQA PRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPN LWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQLVESG GGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYA TYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSNonclinical Pharmacology

[0186] The pharmacological activity of XPAT01 was evaluated across a set of in vitro and in vivo studies. In vitro studies included an assessment of the binding affinity of XPAT01 and XPAT01-TCE with CD3 and EGFR from humans and monkeys and an assessment of the biological activity of XPAT01 and its metabolites in in vitro cell culture systems, includingVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO EGFR-expressing cells and purified human PBMCs. The effect of XPAT01 and XPAT01-TCE on cytokine release from human and monkey PBMCs was also evaluated. The in vivo studies evaluated the efficacy of XPAT01, XPAT01-TCE, and XPATOl-NoClvSite (Table 2) in several mouse xenograft tumor models expressing varying levels of EGFR. The combination of XPAT01 and an anti-PD-1 monoclonal antibody (pcmbrolizumab) in the ovarian SK-OV-3 xenograft tumor model was also evaluated.Table 2. Nomenclature of XPAT01, metabolites, and related moleculesMolecule DescriptionXPAT01 Clinical candidate. Fully masked, protease-activated TCEXPTA01-TCE Fully unmasked TCE. Both XTEN masks removedXPATOl(lx-N) Partially unmasked TCE. N-terminal XTEN remains intact; C-terminal XTEN mask removedXPATOl(lx-C) Partially unmasked TCE. C-terminal XTEN remains intact. N-terminal XTEN mask removedXPATOl-NoClvSite Similar to XPAT01, but lacking protease cleavage sitesPrimary pharmacodynamicsIn vitro pharmacologyCharacterization of binding by XPAT01 and its proteolytic metabolites to human and monkey CD3e and EGFR

[0187] The binding affinity (KD) of XPAT01 and its metabolites to human and cynomolgus monkey CD3e and human and rhesus monkey EGFR at 37°C by surface plasmon resonance was evaluated in Study XPATOl-zra vitro-01. The extracellular domain of rhesus and cynomolgus EGFR share 100% sequence identity. As shown in Table 3, XPAT01 and its metabolites demonstrated comparable binding affinities for both human and monkey EGFR and CD3e. XPAT01 exhibited approximately 20-fold and 2-fold lower affinity to human EGFR and CD3e, respectively, than did XPAT01-TCE. The partially unmasked metabolites XPATOl(lx-N) and XPATOl(lx-C) generally bound to CD3s and EGFR with affinities that were intermediate between XPAT01-TCE and XPAT01.Table 3. Summary of equilibrium binding constants for XPAT01 and its metabolites to human and monkey EGFR and CD3e

[0188] KD ± SE (nM)Test article Human MonkeyEGFR CD3s EGFR CD3EXPAT01 0.736 ± 0.007 57 ± 9 1.048 ± 0.114 167 ± 3Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO XPATO l(lx-N) 0.205 ± 0.002 36 ± 3 0.250 ± 0.02 47 ± 5XPATO l(lx-C) 0.184 ± 0.040 32 ± 1 0.238 ± 0.01 43 ± 2XPATO 1-TCE 0.034 ± 0.003 29.2 + 0.2 0.042 ± 0.012 28 +4Assessment of T-cell-dependent cellular cytotoxicity mediated by XPATO 1 and its proteolytic metabolites against human cancer cell lines and normal primary human keratinocytes

[0189] The pharmacological activity of XPATO 1 and its proteolytic metabolites when incubated in vitro with human PBMCs and EGFR-expressing human cancer cells or normal primary human keratinocytes was evaluated. The primary objective of these studies was to characterize the in vitro cytotoxicity of XPATO 1 and its metabolites against EGFR-expressing human tumor cells and normal primary human keratinocytes. An additional objective was to assess the impact of XPAT01 and its proteolytic metabolites on T-cell activation in the presence of EGFR-expressing cells by quantifying the release of cytokines in culture supernatant and the expression of surface activation markers on CD4+ and CD8+ T cells.

[0190] Human T-cell-mediated in vitro killing was evaluated against human cancer cell lines that expressed increasing levels of EGFR (MCF-7, HT-29, MDA-MB-231, and A-431) and against normal primary human keratinocytes (from 2 independent lots / sources). All human cancer cell lines stably expressed a codon-optimized version of luciferase, Luc2, which was used for study readout and quantification of cytotoxicity. The cell surface receptor density of EGFR on target cell lines was determined using the flow cytometry -based QuantiBrite™ quantitative analysis kit. Surface EGFR expression (receptor density) for MCF-7-Luc2, HT-29-Euc2, MDA-MB-2 1 -Euc2, and A-431 -Euc2 cells, as well as normal primary human keratinocytes, are provided in Table 4.

[0191] Representative in vitro cytotoxicity profiles of XPATO 1 and its metabolites in MCF-7-Luc2, HT-29-Luc2, MDA-MB-231-Luc2, and A-431-Luc2 cells are shown in FIG. 1, and representative cytotoxicity profiles in normal primary human keratinocytes are shown in FIG. 2. A summary of the potency of XPATO 1 and its metabolites against evaluated cell lines using multiple human PBMC donors is shown in Table 4. XPAT01-TCE demonstrated potent cytotoxicity across all cell lines evaluated; in general, XPATO 1-TCE potency positively correlated with EGFR receptor density. The presence of XTEN masks on XPATO 1 reduced overall drug potency compared with XPATO 1-TCE. The level of in vitro cytotoxicity protection was cell line-dependent, ranging from approximately 12-fold in A-431 -Luc 2 toVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO >17 OOO-fold in HT-29-Luc2, demonstrating the significant functional impact of masking by the 2 XTEN polymer chains.

[0192] In general, the partially unmasked metabolites, XPATOl(lx-N) and XPATOl(lx-C), demonstrated activity intermediate to XPAT01 and XPAT01-TCE across all cancer cell lines. In MCF-7-Luc2 and HT-29-Luc2 cancer cells, both XPAT01 and XPATOl-NoClvSite demonstrated similar and limited cytotoxicity. In contrast, XPAT01 induced greater cytotoxicity in A-431-Luc2 and MDA-MB-231-Luc2 cells than did XPATOl-NoClvSite, which suggests that XPAT01 activity may partially be driven by XPAT01 cleavage (unmasking) and activation during the course of the in vitro cytotoxicity assay and thus these cell lines overestimate the potency of XPAT01 in its intact form. Importantly, while XPAT01-TCE exhibited potent cytotoxicity in normal primary human keratinocytes, limited cytotoxicity was observed for both XPAT01 and XPATOl-NoClvSite, consistent with proteases being tightly regulated in normal tissue.Table 4. Summary of ICso values for XPATOl-mediated cytotoxicity against EGFR-expressing human cancer cell lines and normal primary human keratinocytesTest articleHuman cell line EGFR XPAT01 XPAT01( XPAT01(l XPAT01- XPAT01- Cytotoxicity density _ Ix-N) _ x-C) _ TCE NoClvSite protectiona(count / cell) Geometric meanbICso (nM)CancerousMCF-7-Luc2 8464 ND ND ND 0.11 ND >2700-fold HT-29-Luc2 34552 ND 3.5 1.8 0.018 ND >17000-fold MDA-MB-231-Luc2c41 769 1.7 0.10 0.067 0.0088 ND 193-fold A-431-Luc2d916947 0.052 0.009 0.012 0.0042 9.2 12-fold NoncancerousPrimary human 60246 ND 3.4 2.8 0.24 ND >2100-fold keratinocytesca Cytotoxicity protection calculated based on XPAT01 IC50 / XPAT01-TCE IC50. In cases in which cytotoxicity with XPAT01 was not observed, the highest XPAT01 concentration tested was used in place of an IC50.b Based on evaluation using 5 individual human PBMC donors.c Activity results used for determination of MABEL first-in-human starting dosed XPAT01 potency estimate is likely substantially overestimated based on high rate of unmasking(~80% in cellular fraction) following in vitro incubation with A-431-Luc2 cellsVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Characterization of the in vitro cleavage ofXPATOl by human cancer cell lines and normal primary human keratinocytes

[0193] To directly address the hypothesis that XPAT01 may be protcolytically cleaved and activated during the in vitro cytotoxicity experiment, the core of the XPAT01 molecule was labeled with a fluorophore and incubated with HT-29-Luc2, MDA-MB-231-Luc2, and A-431-Luc2 cells, as well as normal primary human keratinocytes, for 48 hours (in the absence of PBMCs). A cellular fraction and media-only fraction was subsequently harvested, and protein lysates were electrophoresed to separate and visualize XPAT01 and proteolytic metabolites (FIG. 3). Consistent with the in vitro cytotoxicity results, cleavage ofXPATOl to XPAT01-TCE, XPATOl(lx-N), and XPATOl(lx-C) was observed in both the media and cellular fraction for A-431-Luc2 and MDA-MB-231-Luc2 assays. Limited to no XPAT01 metabolites were observed when XPAT01 was incubated with media only, HT-29-Luc2, or normal primary human keratinocytes. These data support that XPAT01 is cleaved and activated in the presence of MDA-MB-231-Luc2 and A-431-Luc2 cancer cells to the extent of approximately 20% and 80% relative abundancy for XPAT01-TCE (in the cellular fraction), respectively.

[0194] The determination of the anticipated biological activity of XPAT01 in plasma and tumor for the minimal anticipated biological effect level (MABEL) FIH starting dose calculation is dependent on an accurate estimate of the potency of fully masked XPAT01, XPATOl(lx-N), and XPATOl(lx-C), as well as fully unmasked XPAT01-TCE.Characterization of cytokine induction by XPAT01 and its proteolytic metabolites by human PBMCs incubated with human cancer cell line HT-29-Luc2

[0195] In addition to assessing the effect ofXPATOl and its metabolites on T-cell -mediated cell killing of EGFR-expressing tumor cells, cytokine secretion was measured from the cell-free supernatant collected from the cytotoxicity experiments with HT-29-Luc2 cells to quantify TCE-induced cytokine release.

[0196] As shown in FIG. 4, for select cytokines, incubation of XPAT01-TCE with HT-29-Luc2 cells induced a concentration-dependent increase in cytokine concentration, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-y, IL-ip, IL-2, IL-4, IL-6, IL- 10, MCP-1, and tumor necrosis factor (TNF)-a. Consistent with the in vitroVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO cytotoxicity results, the presence of XTEN masks on XPAT01 significantly reduced cytokine induction. Following incubation with XPAT01 and XPATOl-NoClvSite, there were limited to no cytokines detected, with the exception of IL-6 and MCP- 1 , for which induction was observed at the highest concentrations evaluated. XPATOl(lx-N) and XPATOl(lx-C) demonstrated intermediate cytokine induction compared with XPAT01 and XPAT01-TCE.Characterization of T-cell activation by XPAT01 and its proteolytic metabolites by human PBMCs incubated with human cancer cell line HT-29-Luc2

[0197] To evaluate T-cell activation, XPAT01 and its proteolytic metabolites were assessed for activity in the presence of human PBMCs cocultured with the EGFR-expressing HT-29-Luc2 cell line.

[0198] As shown in FIG. 5, limited to no activation of T cells was observed by XPAT01 in both the CD4+and CD8+T-cell subsets, whereas XPAT01-TCE induced maximum upregulation of CD69, CD25, and PD-1 expression, with median EC50 activation of T cells in the range of 0.031 to 0.099 nM. As anticipated, the metabolites XPATOl(lx-N) and XPATOl(lx-C) generated an intermediate response, and XPATOl-NoClvSite induced limited to no response. T-cell activation was generally observed at concentrations of each test article that closely mirrored the cytotoxicity activity and cytokine induction results for HT-29-Luc2 cells.

[0199] Together with the above in vitro experiments, these results demonstrate that once there is sufficient crosslinking between an EGFR-expressing cell and the T cell, T-cell activation occurs, resulting in potent EGFR target-dependent lysis of target cells and cytokine release from human PBMCs.Cytometric bead array analysis of human cytokines using PBMCs from healthy human donors and naive cynomolgus monkeys

[0200] The potential of XPAT01-TCE and XPAT01 to trigger cytokine release in primary PBMCs derived from 5 healthy human donors and 5 monkeys was evaluated. PBMCs were treated with soluble and wet-coat formats of XPAT01 and XPAT01-TCE, with concentrationdependent responses examined in triplicate at 5-, 50-, and 500-nM concentrations. An anti-CD3 antibody (OKT3 for humans and SP34 for monkeys) and isotype-matched antibodies were used as positive and negative controls, respectively. The levels of IL-2, IL-4, IL-6, IL-Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO 10, TNF-a, and IFN-y in tissue culture supernatants were measured 24 hours after treatment using a cytometric bead array. Direct T-cell-receptor stimulation by anti-CD3 antibodies in both soluble and plate-bound treatment formats resulted in IL-2, IL-4, IL-6, IL- 10, TNF-a, and IFN-y release in all samples, demonstrating that these PBMC samples had the capacity to release cytokines in response to T-cell activation. Similar cytokine trends were observed between human and monkey PBMCs.

[0201] XPAT01 and XPAT01-TCE did not induce IL-2, IL-4, IL-10, TNF-a, or IFN-y for any donors above the level of untreated controls in the wet-coated format. Wet-coated XPAT01 and XPAT01-TCE induced IL-6 release in some, but not all, donors, and was not clearly dose-dependent.

[0202] XPAT01 and XPAT01-TCE did not induce IL- 2 or IL-4 for any donors above the level of untreated controls in the soluble format. Additionally, XPAT01-TCE did not induce IL- 10, TNF-a, or IFN-y for any donors above the level of untreated controls in the soluble format.

[0203] XPAT01-TCE induced IL-6 in some, but not all, donors, and was not clearly dosedependent. Soluble treatment of XPAT0I (500 nM) induced IL- 10 and TNF-a from all donors, and IFN-y cytokine release from 1 donor. Furthermore, XPAT01 induced IL-6 in a dose-dependent manner, and the magnitude of response at 500 nM was higher than that of the anti-CD3 positive control treatment.

[0204] The in vivo relevance of the in vitro IL-6 induction for XPAT01 in soluble format is unknown. Elevated IL-6 levels were not accompanied by corresponding robust increases in TNF-a, IFN-y, and IL-2, which are cytokines commonly associated with IL-6 secretion under conditions of T-cell activation and cytokine-release syndrome (CRS). This effect was also pronounced in the soluble (not wet-coated) format, which is suggestive of an in vitro artifact.In vivo pharmacology

[0205] Several in vivo efficacy studies were conducted to evaluate the impact of XPAT01 in redirecting T cells to kill EGFR-expressing tumors. Because XPAT01 is not cross-reactive to mouse EGFR or CD3, immunodeficient mice were inoculated with human EGFR-expressing xenograft tumors and engrafted with human PBMCs as a source of effector T cells. Antitumor activity of XPAT01 was assessed in the LoVo tumor model (27756 EGFR / cell), the HT-29 tumor model (42 167 EGFR / cell), and the MDA-MB-231 tumor model (74656 EGFR / cell).Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Additionally, a study was performed in the SK-OV-3 tumor model (53 185 EGFR / cell) to evaluate whether antitumor activity was enhanced when XPAT01 was combined with pembrolizumab, a clinically approved therapeutic antibody that blocks PD-1 on lymphocytes.In vivo efficacy study ofXPATOl, XPAT01 -NoClvSite, and XPAT01 -TCE in the treatment of the LoVo human CRC tumor model in PBMC-engrafted NSG mice

[0206] The in vivo efficacy ofXPATOl, XPAT01 -NoClvSite, and XPAT01-TCE was evaluated in the human PBMC-engrafted LoVo CRC tumor model in ODMg-Prkdcsc,dI12rg""; / SzJ (NSG) mice.

[0207] Mice bearing LoVo tumors were randomized into 8 groups of 8 mice. Tumor growth curves between treatment initiation (Day 7) and study termination (Day 27) are shown in FIG. 6. All test articles were well tolerated by the experimental animals, as evidenced by a BWL <8.0% of all treated mice on Day 27 compared with start of treatment for all treated mice. XPAT01-TCE had a potent TGI of 95% (p<.0001) at Day 27 (end of study) compared with vehicle diluent (with PBMCs). Similarly, XPAT01 treatment promoted antitumor activity in a dose-dependent manner. At Day 27, XPAT01 -treated tumors had TGIs of 51% (p=.0585), 81% (p<.0001), 96% (p<.0001), and 106% (p<.0001) at dose levels of 0.5 mg / kg QW, 1.5 mg / kg QW, 3 mg / kg QW, and 3 mg / kg twice weekly (BIW), respectively. Tumors treated with XPAT01 -NoClvSite did not induce TGI (-14%; p= 1.0000) at Day 27 compared with vehicle control (with PBMCs), which suggests that the TGI observed with XPAT01 is dependent on cleavage and removal of the XTEN masks.

[0208] In a separate study, mice bearing LoVo tumors were evaluated for tumor pharmacodynamic assessments of CD4, CD8, and PD-L1 intratumoral status by immunohistochemistry. Mice bearing LoVo tumors were randomized into 9 groups of 6 mice each and administered vehicle diluent (with PBMCs; 2 donors evaluated), 0.5 mg / kg XPAT01, or 3 mg / kg XPAT01 by bolus IV QW. Selected tumors (n=6 mice per timepoint) were harvested 2, 6, and 9 days after study drug administration and processed for CD4, CD8, and PD-L1 immunohistochemistry. The percentage of CD4+, CD8+, and PD-L1+cells (number of CD4+, CD8+, or PD-L1+per total cells per area) were calculated.

[0209] Results demonstrate that XPAT01 promoted CD4+and CD8+T-cell infiltration in a dose- and time-dependent manner (representative data for CD8+cells shown in FIG.7). At Day 9 after initial treatment, XPAT01 dosed at 3 mg / kg resulted in a significant difference inVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO CD4+(4.28%; p=.0007) and CD8+(12.95%; p=.0002) cells compared with tumors treated with vehicle diluent (CD4+, 0.46%; CD8+, 1.13%). There was also a significant difference observed between 0.5 mg / kg XPAT01 and vehicle diluent control in CD8+(4.14% vs 1.13%, respectively; p=.O273) at Day 9. Similarly, a significant difference was observed between 3 mg / kg XPAT01 and vehicle diluent control in PD-L1+cells (2.98% vs 0.45%, respectively; p=.0040) at Day 9. Taken together, these data demonstrate that XPAT01 treatment leads to an influx of CD4+and CD8+cells and upregulation of PD-L1, which is consistent with the MOA of the drug.In vivo efficacy study ofXPATOl, XPAT01 -NoClvSite, and XPAT01-TCE in the treatment of the HT-29 human CRC tumor model in PBMC- engrafted NSG mice

[0210] The in vivo efficacy ofXPATOl, XPAT01 -NoClvSite, and XPAT01-TCE was evaluated in the human PBMC-engrafted HT-29 human CRC tumor model in NSG mice.

[0211] Mice bearing HT-29 tumors were randomized into 7 groups of 6 mice. Tumor growth curves between treatment initiation (Day 4) and study termination (Day 24) are shown in FIG. 8. All test articles were well tolerated by the experimental animals, as evidenced by the similar average body weight gain in that ranged from 6.2% to 14.5% across all experimental groups.

[0212] XPAT01-TCE had a potent TGI of 108% (pc.0001) at Day 24 (end of study) compared with vehicle diluent (with PBMC). Similarly, XPAT01 treatment promoted antitumor activity in a dose-dependent manner. At Day 24, XPAT01 -treated tumors had TGIs of 65% (p=.O593), 73% (p=.0051), and 75% (p=.0005) at dose levels of 0.5, 1, and 3 mg / kg, respectively. Tumors treated with XPAT01 -NoClvSite induced limited TGI (16%; p=1.000) at Day 24 compared with vehicle control (with PBMC), which suggests that the TGI observed with XPAT01 is dependent on cleavage and removal of the XTEN masks.In vivo efficacy study ofXPATOl and XPAT01-TCE in the treatment of human breast tumor MDA-MB-231 cells in PBMC-engrafted NSG mice

[0213] The in vivo efficacy of XPAT01 and XPAT01-TCE was evaluated in the human PBMC-engrafted MDA-MB-231 human breast tumor model in NSG mice.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0214] Mice bearing MDA-MB-231 tumors were randomized into 6 groups of 8 mice each. Tumor growth curves between treatment initiation (Day 10) and study termination (Day 36) are shown in FIG. 9. All test articles were well tolerated by the experimental animals. All treated mice had BWL <7.4% on Day 36 compared with the start of treatment.

[0215] XPAT01-TCE had a potent TCd of 106% (p<.0001) at Day 36 (end of study) compared with vehicle diluent (with PBMCs). Similarly, XPAT01 treatment promoted antitumor activity in a dose-dependent manner. At Day 36, XPAT01 -treated tumors had TGIs of 50% (p=.5198), 91% (p<.0001), and 114% (p<0001) at dose levels of 0.1 mg / kg, 0.5 mg / kg, and 2 mg / kg, respectively.In vivo efficacy study of the combination ofXPATOl and pembrolizumab in the treatment of human ovarian tumor SK-OV-3 in PBMC-engrafted NSG mice

[0216] The in vivo efficacy of XPATOl and pembrolizumab (anti-PD-1) was evaluated in the human PBMC-engrafted SK-OV-3 human ovarian tumor model in NSG mice.

[0217] Pembrolizumab, an immune checkpoint inhibitor, is a monoclonal antibody that binds to PD-1 on lymphocytes and blocks its interaction with its ligands (PD-L1 and PD-L2) on tumor cells. Inhibiting the negative immune regulation caused by PD-1 receptor signaling can induce antitumor responses.

[0218] Mice bearing SK-OV-3 tumors were randomized into 10 groups of 8 mice. Tumor growth curves between treatment initiation (Day 19) and study termination (Day 29) are shown in FIG. 10. Both test agents (XPAT01 and pembrolizumab) were generally well tolerated by test animals, as all groups had a mean body weight gain in the range of 2.9% to 9.8% on Day 29 compared with Day 19.

[0219] XPAT01-TCE had a TGI of 41.5% (p=.OO86) at Day 29 (end of study) compared with vehicle diluent (with PBMCs). XPAT01 treatment promoted antitumor activity in a dosedependent manner. At Day 29, XPAT01 -treated tumors had TGIs of 34.6% (p=.1192), 27.8% (p=.l 192), 52.4% (p=.0007), and 58.2% (p<0001) at dose levels of 0.01 mg / kg QW, 0.1 mg / kg QW, 0.5 mg / kg QW, and 2 mg / kg QW, respectively. Tumors treated with XPAT01-NoClvSite did not induce TGI (-16.5%; p=.7298) at Day 29 compared with vehicle control (with PBMC), which suggests that the TGI observed with XPAT01 is dependent on cleavageVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO and removal of the XTEN masks. Pembrolizumab monotherapy had a TGI of 30.4% (p=.394O) at Day 29 compared with vehicle control.

[0220] The combination of 0.1 mg / kg XPAT01 and 10 mg / kg pembrolizumab exhibited significantly greater TGI (67.9%) than either single agent alone (0.1 mg / kg XPAT01 TGI: 27.8%; p<.0001 and 10 mg / kg pembrolizumab TGI: 30.4%; pc.OOOl).Pharmacokinetics and Product Metabolism In Animals

[0221] The PK of XPAT01 following a single bolus administration was evaluated in healthy and tumor-bearing mice. The TK of XPAT01 following QW IV infusions was evaluated in monkeys as part of the GLP-compliant toxicology study. The metabolism and peripheral stability of XPAT01 was assessed in vitro in plasma samples from humans (healthy and cancer patients), healthy monkeys, and mice (healthy and tumor-bearing), and in vivo in tumor-bearing mice.

[0222] XPAT01 PK was characterized in mice and monkeys to characterize the clearance of XPAT01 by proteolytic metabolism and nonspecific elimination. The studies in healthy (nontumor-bearing) animals may not fully characterize the impact of EGFR binding on XPAT01 disposition in cancer patients.Single-dose pharmacokinetics in mice

[0223] The PK of XPAT01 was evaluated in a subcutaneous HT-29 human colon cancer model and non-tumor-bearing female NSG mice. A total of 9 HT-29 tumor-bearing mice and 9 control non-tumor-bearing mice were administered a single bolus IV injection of 0.2 mg / kg XPAT01. An additional 9 HT-29 tumor-bearing mice and 9 control non- tumor-bearing mice were administered a single bolus IV injection of 2 mg / kg XPAT01.

[0224] Plasma samples were collected from 3 tumor-bearing and 3 control non-tumor-bearing mice each per dose level prior to IV administration and from 0.5 to 168 hours postdose for determination of XPAT01 concentrations by a qualified ligand-binding assay. PK parameters were determined by noncompartmental analysis using a composite profile and are presented in Table 5.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Table 5. Pharmacokinetic parameters of XPAT01 following a single 0.2 mg / kg or 2 mg / kg intravenous bolus administration in tumor-bearing and control mice XPAT01 dose level and mouse model .. 0.2 mg / kg HT- 0.2 mg / kg 2 mg / kg HT-29 2 mg / kg Control 29 tumor- Control non- tumor- non- tumorbearing tumor-bearing bearing bearing tmax(h) 2 0.5 0.5 0.5 Cmax(nM) 26 25.5 228 204 Cmax / dose (nM / [mg / kg]) 130 128 114 102 tjast(h) 168 168 168 168 AUCiast(nM »h) 1260 1190 11 700 12000 AUCi^ / dose (nM • , ,r„ , 6300 5950 5850 6000 h / [mg / kg]) AUCaAnM’h) 1310 1300 12400 13000 CL (mL / h / kg) 1.08 1.09 1.15 1.09 Vss(mL / kg) 60 74.8 67.1 71.3 ti / 2 (h) 39.7 48.9 41.9 46.1MRToo (h) 55.4 68.3 58.6 65.2

[0225] XPAT01 plasma concentration was not quantifiable in samples collected prior to IV administration but was quantifiable from the first sample collected post-dose (30 minutes) through the last sample collected at 168 hours post-dose in both tumor-bearing and control mice at both XPAT01 dose levels. Following IV bolus administration, XPAT01 plasma concentration generally declined in a monophasic manner in both tumor-bearing and control non-tumor-bearing mice. XPAT01 plasma concentration declined at a similar rate in tumorbearing mice and control non-tumor-bearing mice at both dose levels evaluated. Following IV bolus administration, XPAT01 Cmax and total systemic exposure (area under the plasma concentration-time curve [AUG] from time zero to time of last measurable concentration [AUCiast] and AUG from time zero to infinity | Al IG, ]) were similar in tumor-bearing mice and control non-tumor-bearing mice. Cmax and total system exposure increased with dose in a dose-proportional manner between 0.2 and 2 mg / kg. Volume of distribution at steady state (Vss), clearance, and ti / 2 were similar across XPAT01 dose levels and between the tumorbearing and control non-tumor-bearing mice. Across all 4 groups, XPAT01 ti / 2 was approximately 2 days, ranging from 39.7 to 48.9 hours (Table 5).Single-dose toxicokinetics in cynomolgus monkeys

[0226] The TK of XPAT01 was evaluated in naive male monkeys as part of the non-GLP toxicology Study AX22-01. In this study, XPAT01 was administered by a single 30-minute IV infusion at dose levels of 1.5, 3, 4.5, 8, and 10 mg / kg (single animal per dose level). Serial plasma samples were collected prior to infusion and at multiple timepoints up to 168 hoursVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO following infusion. The concentration of XPAT01 in each plasma sample was quantified using a qualified bioanalytical method.

[0227] TK parameters of XPAT01 were determined following single-dose administration and are presented in Table 6.Table 6. Toxicokinetic parameters following single intravenous administration of 1.5, 3, 4.5, 8, and 10 mg / kg XPAT01 in naive male cynomolgus monkeys (Study AX22-01)XPAT01 dose level (N=l / group)alaili i 1.5 mg / kg3 mg / kg 4.5 mg / kg 8 mg / kg10 mg / kg tmax(h) 0.083 0.083 0.083 0.083 0.083 Cmax(nM) 235 577 1050 1390 2090 CIIlax / dose (nM / [mg / kg]) 157 192 233 174 209 tlast(h) 144 144 144 168 168 AUCiast(nM«h) 5380 12900 30 300 55 200 70300 AUCias / dose (nM • h / [mg / kg]) 3590 4300 6730 6900 7030 AUCV tnM’h) 5730 13 200 31 200 55 900 72 100 CL (mL / h / kg) 1.86 1.61 1.02 1.02 0.987 Vss(mL / kg) 79.5 49.0 33.3 37.2 41.9 t1 / 2(h) 43.6 31.7 31.7 25.5 32.1MRToo (h) 42.7 30.4 32.5 36.6 42.5

[0228] XPAT01 plasma concentrations generally declined in a monophasic manner following a single 30-minute IV infusion in male monkeys, with a ti / 2 ranging from 25.5 to 43.6 hours across the dose range evaluated. XPAT01 plasma concentrations were quantifiable in the first sample collected postinfusion (5 minutes) through the last sample collected (144 or 168 hours) across all dose levels. XPAT01 exposure (Cmax and AUG) increased with increasing dose across the dose range evaluated. At the highest dose level evaluated (10 mg / kg), XPAT01 Cmax was 2090 nM and AUG, was 72 100 nM • h. As each dose level was only evaluated in a single animal, the dataset was not sufficient to evaluate dose-proportionality. The volume of distribution at steady state was similar to plasma volume, ranging from 33.3 to 79.5 mL / kg. Clearance slightly increased with dose, ranging from 0.987 to 1.86 mL / h / kg.Repeat-dose toxicokinetics in cynomolgus monkeysA 4-week repeat-dose (once-weekly) non-GLP toxicokinetics study in naive cynomolgus monkeys

[0229] The TK of XPAT01 was evaluated in naive monkeys as part of the non-GLP toxicology Study AX22-03. In this non-GLP study, XPAT01 was administered by QW IV 60-Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO minute infusions of 0, 0.5, 2, or 4 mg / kg (1 male and 1 female animal / group) for a total of 4 doses. XPAT01 plasma concentrations were quantified using qualified bioanalytical methods.

[0230] Individual TK parameters of XPAT01 were determined following dose administration on Day 1 and Day 15 and are presented in Table 7. Individual XPAT01 plasma concentration-time profiles for the non-GLP toxicology Study AX22-03 are presented in FIG. 12.Table 7. Individual animal XPAT01 toxicokinetic parameters following 4-weekly 60-minute IV infusions of XPAT01 in male and female cynomolgus monkeysXPAT01 dose level and sexParameter Day 0.5 mg / kg 2 mg / kg 4 mg / kgF M F M F Mtmax(h) 1 6 0.083 0.083 0.083 0.083 0.08315 0.083 0.083 0.083 0.083 0.083 6 Cmax (nM) 1 72.3 67.3 321 231 785 77315 76.7 74.6 105 97.9 125 304 Cmax / dose (nM / [mg / kg]) 1 145 135 161 116 196 19315 153 149 52.5 49.0 31.3 76.0 tlast (h) 1 96 168 168 168 168 16815 168 48 6 6 6 168 AUCiast (nM’h) 1 2320 2100 11 900 6160 21 400 22 10015 3140 1550 492 402 476 16300 AUCiast / dose 1 4640 4200 5950 3080 5350 5530 (nM’h / [mg / kg]) 15 6280 3100 246 201 119 4080 CL (mL / h / kg) 1 1.48 1.63 1.18 2.28 1.33 1.2915 1.10 2.23 NR NR NR 1.74 Vss (mLAg) 1 38.6 62.4 42.6 64.7 30.1 35.015 46.6 31.3 NR NR NR 78.4 ti / 2 (h) 1 20.6 42.6 30.5 26.8 16.3 16.215 32.1 9.83 NR NR NR 21.0 Accumulation ratio 15 1.35 0.738 0.0413 0.0653 0.0222 0.738

[0231] In this non-GLP study, following a 60-minute IV infusion on Day 1, XPAT01 plasma concentration was quantifiable through 168 hours postdose in 5 of the 6 animals administered XPAT01, and through 96 hours postdose in the female animal administered 0.5 mg / kg XPAT01. Following administration of XPAT01 on Day 1, XPAT01 plasma concentration declined, with a terminal ti / 2 ranging from 16.2 to 42.6 hours. XPAT01 exposure (Cmax and AUG) increased with increasing dose in a generally dose-proportional manner across the dose range evaluated and was generally similar between the male and female animals. However, because each dose level was only evaluated in a single male and a single female, doseproportionality and sex effect could not be sufficiently evaluated to draw conclusions. At the highest dose level evaluated (4 mg / kg), XPAT01 Day 1 Cmax was 785 and 773 nM in the female and male animals, respectively, and XPAT01 Day 1 AUCiast was 21 400 and 22 100Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO nM «h in the female and male animals, respectively. XPAT01 clearance on Day 1 was similar across the dose levels evaluated, ranging from 1.18 to 2.28 mL / h / kg.

[0232] Following repeat IV infusion, XPAT01 exposure was generally lower following administration on Day 15 than following administration on Day 1. After a 60-minute IV infusion on Day 15, XPAT01 plasma concentration was quantifiable through 168 hours following administration on Day 15 in only 2 animals (female animal administered 0.5 mg / kg XPAT01 and male animal administered 4 mg / kg XPAT01). The accumulation ratio, based on AUCiast, for these 2 animals ranged from 0.738 to 1.35. In contrast, XPAT01 plasma concentration was only quantifiable through 6 hours following administration on Day 15 in 3 animals. The accumulation ratio for these 3 animals ranged from 0.0222 to 0.0653.A 4-week repeat-dose (once-weekly) GLP toxicokinetics study in naive cynomolgus monkeys

[0233] Additionally, the TK of XPAT01 was evaluated in naive monkeys as part of the GLP toxicology Study. In this GLP study, XPAT01 was administered by QW IV 1-hour infusions of 0, 0.15, 0.5, or 1.5 mg / kg (one-half were male and one-half were female animals; n=6 / group for dose <0.5 mg / kg and n=10 for the 1.5- and 0-mg / kg groups) for a total of 4 doses. XPAT01 and XPAT01-TCE plasma concentrations were quantified while using validated bioanalytical methods. Seram samples were also tested for the presence of AD As while using a validated method.

[0234] Mean TK parameters of XPAT01 were determined following dose administration on Day 1 and Day 22 for GLP toxicology study and are presented in Table 8. Mean XPAT01 plasma concentration- time profiles at Days 1 and 22 for GLP toxicology Study AX23-01 are presented on a semilogarithmic scale in FIG. 13.Table 8. XPAT01 toxicokinetic parameters following repeat weekly intravenous administration in male and female cynomolgus monkeysXPTA01 dose level and sexa0.15 mg / kg 0.5 mg / kg 1.5 mg / kg „ F (N=3) M (N=3) F (N=3) M (N=3) F (N=3) M (N=3) Parameter Day 1 1.08 1.08 1.08 1.08 1.08 1.08 (2; 1.08- (3; 1.08- (3; 1.08- (3; 1.08- (5; 1.08- (5; 1.08-tmax (h)1.08) 1.08) 7.00) 1.08) 1.08) 1.08)22 1.08 1.08 1.08 1.08 1.08 1.08Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO (3; 1.08- (3; 1.08- (3; 1.08- (3; 1.08- (5; 1.08- (5; 1.08- 1.08) 1.08) 1.08) 1.08) 1.08) 1.08) 1 21.8 (2, NA) 19.9 (3, 47) 80.5 (3, 13) 77.8 (3, 5) 248 (5, 14) 256 (5, 11) Cmax (nM)2222.4 (3, 8) 14.7 (3, 57) 23.3 (3, 89) 55.0 (3, 62) 89.6 (5, 91) 36.3 (5, 48) AUCiesh (nM •1 883 C2’NA)684 f3'41)2090(3>9)2390<3’14)6740 9 | 7430I5’13) h) 22 563 (3, 44) 438 (2, NA) NA 2710 (2, NA)2960 (1, NA) NA Terminal ti / 2 (h) 1 37.0 (3, 11) 39.0 (1, NA) 36.7 (3, 7) NA 37.7 (5, 4) 34.2 (4, 8) 122 45.1 (1, NA) NA NA NA NA NA Accumulation 22 / 1 0.478 (2, 1.08 (2, NA) NA 1.16 (2, NA) 0.395 (1, NA Ratio (AUCiesh)bNA) NA)N = number of animals treated; n = number of animals with evaluable TK parameters.aValues are means (n, CV) except for tmax, which is presented in hours following the beginning of infusion and reported as median (n; range).bAccumulation ratio (AUCT68h) = Day 22 AUCiesh / Day 1 AUCiesh.

[0235] Following IV infusion on Day 1, XPAT01 plasma concentration declined slowly in a biphasic manner. The slow decline in plasma concentration suggests that XPAT01 was stable in systemic circulation and not rapidly converted to proteolytic metabolites. Both Cmax and total systemic exposure (AUG from time zero to 168 hours [AUCiesh]) generally increased in a dose-proportional manner between 0.15 and 1.5 mg / kg. In the recovery phase, XPAT01 plasma concentration declined relatively slowly in all dose groups, with a ti / 2 of approximately 36 hours.

[0236] At the highest dose level evaluated (1.5 mg / kg), mean XPAT01 Day 1 Cmax was 252 nM (n=10), and XPAT01 Day 1 AUCiesh was 7085 nM • h (n=10) when combining female and male groups. PK parameters were similar between female and male animals at each dose level.

[0237] Following repeat IV infusion, XPAT01 exposure was generally lower following administration on Day 22 than following administration on Day 1. After a 60-minute TV infusion on Day 22, XPAT01 plasma concentration was quantifiable through 168 hours postdose in only 1 animal administered 0.15 mg / kg XPAT01 and 1 animal administered 0.5 mg / kg XPAT01, and was quantifiable through 7 hours postdose in 8 of the 10 animals administered 1.5 mg / kg XPAT01. There was no accumulation of XPAT01 observed at all dose levels based on an accumulation ratio of AUCiesh on Day 22 relative to Day 1.

[0238] AD As to XPAT01 were detected in most animals in all groups. All ADA-positive animals exhibited a rapid decline in XPAT01 plasma concentration upon repeat dosing. TheVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO mean accumulation ratio based on AUCiesh on Day 22 ranged from 0.395 to 1.16, indicating significant exposure reduction potentially due to ADA generations.Metabolism

[0239] The formation of the partially masked metabolites XPATOl(lx-N) and XPAT01(lx-C) and the fully unmasked proteolytic metabolite XPAT01-TCE was evaluated upon ex vivo incubation of a fluorescently labeled XPAT01 in plasma, and upon administration of a fluorescently labeled XPAT01 in tumor-bearing mice. The plasma concentration of fully unmasked XPAT01-TCE was also evaluated in monkeys administered XPAT01 for the GLP toxicology study.Ex vivo stability ofXPATOl in human, cynomolgus monkey, and mouse plasma

[0240] The extent of unmasking of XPAT01 in plasma collected from healthy human adults and patients with cancer was characterized and compared with the stability of XPAT01 in plasma collected from monkeys, as well as healthy and tumor-bearing NSG mice. These ex vivo data were used to estimate the stability of XPAT01 in circulation and to assess the relevance of the animal model systems used to evaluate the nonclinical safety and efficacy of XPAT01. However, because this is a closed system with no clearance mechanisms, it will overestimate the accumulation of cleavage products.

[0241] The relative amount of XPAT01 (measured as a percentage) spiked into plasma samples slowly declined over time in all plasma samples upon incubation at 37°C, resulting in an increase in the level of the partially and fully cleaved metabolites.

[0242] After incubation at 37°C for 3 days, the median relative amount of XPAT01 ranged from 85% to 88% across the groups evaluated, which suggests that XPAT01 is largely stable in plasma. In addition, the median relative amount of the fully unmasked form of XPAT01 ranged from 0.5% to 0.9% across the various groups evaluated, which suggests that, if cleavage to XPAT01-TCE in plasma occurs in vivo, XPAT01-TCE would form slowly over time. The rate of cleavage and relative amounts of XPAT01 and its cleavage products were negligible and generally similar across the different groups evaluated, indicating that the peripheral stability ofXPATOl is similar in plasma from humans, monkeys, and tumorbearing mice, and which suggests that there is low risk of cleavage in systemic circulation.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Evaluation of the levels ofXPATOl and metabolites in plasma and tissues in tumor-bearing mice

[0243] The in vivo unmasking ofXPATOl (Scy5.5) and XPAT01 (Scy7.5), 2 fluorescently labeled derivatives of XPAT01, was evaluated at 2 days after injection in human cell line-derived NSG mouse models in Studies AX22-50A (6 mice in the MDA-MB-231 model), AX22-50C (4 mice in the LoVo model), AX22-50D (4 mice in the HT-29 model) and AX22-50F (4 mice in the A -431 model).

[0244] The mean concentration of XPAT01 and its metabolites across the tissues were tested. Concentrations of XPAT01-TCE were below the limit of quantification for all normal tissue and plasma, with the exception of 1 mouse in which XPAT01-TCE was quantified in plasma. The relative level of XPATOl(lx-N), XPATOl(lx-C), and XPAT01-TCE in each of the tissues was evaluated. Analysis of tumor homogenates revealed an average of 9.9% XPAT01-TCE in tumors relative to XPAT01 and its derived metabolites. Based on the assay LLOQ, the concentration of XPAT01-TCE was less than 0.1% that of XPAT01 in all but one of the plasma samples tested. These data suggest that the cleavage of XPAT01 primarily occurs in tumors and that low levels of XPAT01 metabolites are formed in the peripheral tissues or in plasma. In addition, the differences in the relative abundance profile of XPAT01 metabolites between tumors and peripheral tissues and plasma suggest that once the metabolites of XPAT01 are formed in the tumor, they do not appear to leak into the systemic circulation or peripheral tissues.Evaluation of the concentration ofXPATOl-TCE in plasma in cynomolgus monkeys administered XPAT01

[0245] The plasma concentration of XPAT01-TCE in male and female monkeys administered XPAT01 QW for 4 weeks was measured as part of the GLP toxicology study.

[0246] XPAT01-TCE concentrations were below the assay limit of quantification (<0.400 nM) in all samples following the first administration ofXPATOl on Day 1 in all animals. Following repeat dosing, the XPAT01-TCE concentration was quantifiable in 2 samples collected following the fourth dose on Day 22 from 1 of 10 animals in the 1.5-mg / kg group, the highest dose tested. The reported 2 quantifiable XPAT01-TCE concentrations were 0.883 nM (5 minutes post-end of infusion [EOI] on Day 22) and 0.687 nM (6 hours post-EOI onVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Day 22). These data indicate that XPAT01 is largely stable in systemic circulation and that systemic exposure to the fully unmasked metabolite XPAT01-TCE is minimal.Nonclinical Toxicology

[0247] The nonclinical safety evaluation program for XPAT01 was designed in consideration of ICH S6, Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals, and S9, Nonclinical Evaluation for Anti cancer Pharmaceuticals. All toxicology studies required to be conducted in compliance with GLP regulations were conducted in compliance with GLPs.

[0248] XPAT01 is not cross-reactive with rodent EGFR or CD3. The nonhuman primate (NHP) was considered a relevant species for the assessment of toxicity of XPAT01 based on similar binding affinities between humans and monkeys for both CD3 and EGFR, and all in vivo toxicology studies were performed in monkeys.

[0249] The toxicological profile of XPAT01 was assessed in preliminary single- and repeatdose non-GLP studies, and in a 1 -month repeat-dose GLP toxicology study with a 4- week recovery period. XPAT01 was assessed via IV infusion at doses up to 10 mg / kg. Endpoints assessed included TK, clinical pathology, ophthalmology, ECG, ADA levels, cytokine levels, immunophenotyping, and histopathology. Consistent with ICH S6, no stand-alone safety pharmacology studies or genotoxicity studies were conducted; instead, safety pharmacology endpoints were incorporated into the GLP study.

[0250] Additionally, XPAT01-TCE, the fully unmasked metabolite, and the partially unmasked proteolytic metabolites XPATOl(lx-N) and XPATOl(lx-C) were also evaluated in single-dose non-GLP studies.Dose range finding study ofXPATOl and XPAT01-TCE in naive cynomolgus monkeys

[0251] The objective of this exploratory non-GLP study was to determine the potential toxicity ofXPATOl when administered to monkeys as a single 30-minute IV infusion. In addition, the systemic exposure of XPAT01 and plasma cytokine levels were determined. The study design is described in Table 9.Table 9. Study AX22-01 single-dose study designA . ni .ma ,l _ ID_ S „ex 1 est article D .ose , l,e ,ve ,l Do .seTvolume ConcentrTa .tion _ Dosi .ng rout ,eb(mg / Kg) (mL / Kg) (mg / mL)a” EC994 M XPAT01 1.5 10 0.15 IV MB1059 M XPAT01 3 10 0.3 IVVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO SB640 M XPAT01 4.5 10 0.45 IV SBZ492 M XPAT01 10 10 1 IV UZ1060 M XPAT01 8C10 0.8 IV a Test article for <4.5-mg / kg dose levels formulated in 20 mM citrate, 10% trehalose, and 0.02% PS-80; pH 5.7. Test article for >8-mg / kg dose levels formulated in 20 mM histidine and 154 mM sodium chloride; pH 6.5. Test articles were diluted in 0.9% (w / v) sodium chloride to achieve the desired concentration. Bulk test article was provided at a concentration of 0.45 mg / mL for the animals dosed at <4.5 mg / kg and 1.28 mg / mL for the animals dosed at >8 mg / kg.b 30-minute infusion.c The animal that was dosed at the 8-mg / kg dose level was originally planned to receive 4 weekly doses, but dosing was stopped after a single dose administration due to poor tolerability of the test article and the animal was euthanized on Day 8.d

[0252] Naive male monkeys (between 2 and 3 years of age at the initiation of the dosing) were included in the study. The animals received a single administration of an aqueous solution of XPAT01 at 1.5, 3, 4.5, 8, or 10 mg / kg by a 30-minute IV infusion and were evaluated up to 168 hours post-EOI. The animal that received the 8-mg / kg dose was originally planned to receive 4 weekly doses, but dosing was stopped after a single dose administration due to poor tolerability of the test article, and the animal was euthanized on Day 8.

[0253] The parameters evaluated included mortality, clinical observations, body temperature, body weight, qualitative food consumption, clinical pathology (hematology and clinical chemistry), bioanalysis, ADA formation, TK, cytokine analysis, organ weights, and macroscopic and microscopic observations. For the animals that received a single administration of XPAT01 at 1.5, 3, 4.5, or 10 mg / kg, plasma samples were collected prior to infusion and at 5 minutes and 2, 6, 24, 48, 96, 144, and 168 hours post-EOI. For the animal that received a single administration of XPAT01 at 8 mg / kg, plasma samples were collected prior to infusion and at 5 minutes and 2, 6, 24, 48, 96, and 144 hours post-EOI. Cytokines (GM-CSF, IFN-y, IL- 1 , IL-2, IL-4, IL-6, IL- 10, MCP-1, and TNF-a) and TK were evaluated from the plasma samples collected at various timepoints as described above. Blood samples for hematology and clinical chemistry were collected prior to dosing and 24 hours post-EOI. Histopathology was performed on animals dosed once with XPAT01 at 1.5, 8, and 10 mg / kg on Days 10, 8, and 8, respectively.

[0254] A single-dose administration of XPAT01 at 1.5, 3, or 4.5 mg / kg was well tolerated. Slight increase in body temperature and loose stools were observed in the animals treated atVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO >1.5 mg / kg XPAT01. XPAT01 -related changes in hematology and serum chemistry were a slight to moderate decrease in lymphocytes and monocytes: an increase in neutrophils (4.5 mg / kg); a mild to marked increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT); and moderate increases in CRP, creatine kinase (CK), and ferritin. These clinical pathology changes were without microscopic correlates at the 1.5-mg / kg dose level (animals at the 3- and 4.5-mg / kg dose levels were not examined microscopically). By contrast, XPAT01-TCE at 0.15 mg / kg caused transient poor appetite (day 2), loose stool (day 5) and PD modulation (e.g., elevated AST / ALT). Therefore, compared to the unmasked TCE core, XPAT01-TCE, XPAT01 provides a >~250x safety margin.

[0255] Single-dose administration of 8 and 10 mg / kg XPAT01 was not tolerated and induced clinical findings, including loss of appetite, BWL, and persistent loose tools. Hematology and serum chemistry changes included elevations in AST, ALT, bilirubin, CRP, CK, and ferritin and decreases in lymphocytes, monocytes, hemoglobin, hematocrit, and red blood cells.

[0256] In the non-clinical study, XPAT01-TCE exhibited a ~20-fold faster clearance than XPA01 (FIG. 14A), while administration of both molecules led to dose-dependent cytokine (e.g., IL-6) induction (FIG. 14B).

[0257] Microscopically, all XPAT01 -treated animals evaluated had minimal to mild increased germinal centers in lymph nodes, minimal increased mitoses in lymphocytes within lymph nodes, and a mild expansion of the paracortex. Minimal to mild skeletal muscle degeneration was noted at >8 mg / kg. Additional changes noted were moderate hepatocellular vacuolation in the animal dosed once with 8 mg / kg XPAT01 and marked ulceration of the cecum in the animal dosed once with 10 mg / kg XPAT01.

[0258] Increases in MCP-1 and IL-6 were observed after administration of XPAT01 at all dose levels. The increases in MCP-1 and IL-6 were highest with 10 mg / kg XPAT01. Peaks were observed at 6 hours post-EOI, with decreasing levels 12 hours post-EOI. At dose levels of XPAT01 <8 mg / kg, although quantifiable increases were observed, the values did not demonstrate any dose dependence. XPAT01 -related increases in other cytokines monitored (GM-CSF, IFN-y, IL-1 [3, IL-2, IL-10, and TNF-a) were observed only with the high-dose levels (8 and / or 10 mg / kg) of XPAT01. Peak was observed between 0.5 and 6 hours, returning close to baseline 24 hours post-EOI.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0259] A single dose of 4.5 mg / kg was considered to be the maximum tolerated dose (MTD) ofXPATOl.Repeat-dose toxicityA 4-week repeat-dose (once weekly) toxicity study ofXPATOl and single-dose study of XPAT01( Ix-N) and XPATOl(lx-C) administered by IV infusion in naive cynomolgus monkeys

[0260] The objective of this exploratory non-GLP study was to determine the potential toxicity and characterize the TK profile of XPAT01 when administered to monkeys as a 60-minute IV infusion QW for 4 weeks. In addition, the study included a characterization of ADA responses and assessment of XPAT01 -related changes in cytokines.

[0261] Naive male and female monkeys (between 2 and 3 years of age at the initiation of the dosing) were included in the study. The animals received weekly administrations of an aqueous solution of vehicle or XPAT01 (formulated in 20 mM citrate, 8% trehalose, and 0.02% PS-80; pH 6.3 and diluted 0.9% [w / v] sodium chloride for injection) at 0.5, 2, or 4 mg / kg by a 60-minute IV infusion.

[0262] The parameters evaluated included mortality, clinical signs, body temperature, body weight, qualitative food consumption, clinical pathology (hematology, clinical chemistry, and coagulation), organ weights, macroscopic and microscopic observations, ADA formation, TK, cytokine analysis, and immunophenotyping. Blood samples for TK, ADA and cytokine (GM-CSF, IFN-y, IL-10, IL-2, IL-4, IL-6, IL-10, MCP-1, and TNF-a) evaluation were collected on Day 1 prior to infusion and 6 and 24 hours post-EOI, and on Days 15 and 22 prior to infusion and 24 hours post-EOI. Blood samples for hematology, clinical chemistry, and coagulation were collected on Days 1 and 22 prior to dosing and 24 hours post-EOI. Blood samples for immunophenotyping were collected predose and 24 hours post-EOI on Days 1, 15, and 22. Animals were necropsied on Day 29. Macroscopic observations were recorded and select tissues were collected for organ weights and histopathology evaluation.

[0263] Weekly administration of XPAT01 at 0.5, 2, and 4 mg / kg for 4 weeks was tolerated. All animals survived to the scheduled necropsy on Day 29. XPAT01 -related clinical observations were limited to loose stool / diarrhea in the female animal at 4 mg / kg. The male animal at 4 mg / kg exhibited decreased appetite on multiple days from Day 9 through Day 20 and had a 14% decrease in body weight on Day 29. The female animal at 0.5 mg / kg also hadVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO poor appetite on Day 9 continuing throughout the study with no effect on body weight. Body temperature elevations were observed at >2 mg / kg XPAT01.

[0264] XPAT01 -related changes in clinical pathology parameters were observed in all XPAT01 -treated animals on Day 1, 24 hours post-EOI, and included increases in AST, ALT, CRP, and CK, as well as decreases in lymphocytes and monocytes. At the 4-mg / kg dose level, increases in CRP and CK were also observed on Day 22, 24 hours post-EOI, and increases in ferritin were observed after the Day 1 and Day 22 dose administrations. Relative to the vehicle control group and prcdosc values, increases in prothrombin time were observed in the male animal treated with 2 mg / kg XPAT01 and the female animal treated with 4 mg / kg XPAT01. An increase in activated partial thromboplastin time (PTT) was observed in the male animal treated with 4 mg / kg XPATO 1. An increase in fibrinogen was observed in the male animal treated with 4 mg / kg XPATO 1 on Day 23 after the fourth dose.

[0265] XPATO 1 -related macroscopic observations were limited to mildly enlarged spleens in the male animals at >2 mg / kg with accompanying increases in spleen weight relative to body weight.

[0266] XPATO 1 -related microscopic changes were limited to the lymphoid organs (spleen, gut-associated lymphoid tissue, and mandibular, mesenteric, and draining lymph nodes) and were characterized by minimal to mild increased lymphoid follicles, paracortical hyperplasia, and increased mitosis in the lymph nodes. Additionally minimal increases in myeloid cells were present in the bone marrow of some treated animals. The male animals in general had a slightly higher degree of severity for histopathology findings than did female animals.

[0267] Administration of XPAT01 resulted in decreases of total T-lymphocyte-relative percentages on Day 1, 24 hours post-EOI and on Day 15, 24 hours post-EOI, for the animals treated with 2 and 4 mg / kg XPATO 1. No decrease was observed on Day 22, 24 hours post-EOI. Relative to the vehicle control group, an increase in CD69+T-cytotoxic lymphocytes was observed on Day 1, 24 hours post-EOI, in all animals and on Day 22, 24 hours post-EOI, in the male animal treated with 4 mg / kg XPATO 1. Limited XPATO 1 -related changes were observed in the CD25+T-cytotoxic lymphocyte population. An increase in CD69+T-helper lymphocytes was observed in the female animal treated with 4 mg / kg XPATO 1 on Day 1, 24 hours post-EOI. An increase in CD25+T-helper lymphocytes was observed in the groups treated with 2 and 4 mg / kg XPATO 1 on Day 1, 24 hours post-EOI.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0268] Administration of XPAT01 was associated with a transient, dose-dependent elevation in most of the cytokines evaluated (IL-ip, IL-2, IL-6, IL-10, IFN-y, MCP-1, and TNF-a). The most pronounced effect was observed with IL-6 and MCP-1, for which peak concentrations were elevated over 100-fold above that observed in the vehicle control animals. Serum cytokines were generally not elevated following administration of vehicle or 0.5 mg / kg XPAT01. In general, cytokine concentrations peaked at 6 hours following the first dose on Day 1 and quickly returned to or near baseline by 24 hours following the first dose. Cytokine levels were not elevated prior to or 24 hours following administration on Days 15 and 22.A 1 -month GLP toxicology study ofXPATOl in cynomolgus monkeys followed by a 4-week recovery

[0269] The objectives of this GLP study were to determine the potential toxicity of XPAT01 when given by IV infusion (over 60 minutes) QW for 4 weeks in monkeys and to evaluate the potential reversibility of any toxicological findings following a 4-week recovery.

[0270] A total of 32 male and female monkeys were included in the study. Animals received 4 QW administrations ofXPATOl at 0, 0.15, 0.5, or 1.5 mg / kg by 60-minute IV infusion on Days 1, 8, 15, and 22.

[0271] The following parameters and endpoints were evaluated in this study: mortality, clinical observations, body weights, appetence, body temperature, local irritation assessments, neurological examinations, respiratory rate, ophthalmology, ECG, clinical pathology (hematology, coagulation, clinical chemistry [including ferritin], and urinalysis), TK, ADA analysis, immunophcnotyping, cytokine analysis, organ weights, and macroscopic and microscopic examinations.

[0272] There were no unscheduled deaths over the course of the study. Repeated IV infusion of 0.15, 0.5, or 1.5 mg / kg / doscs ofXPATOl resulted in no changes or effects in clinical observations, body weights, appetence, body temperature, local irritation assessments, neurological examinations, respiratory rate, ophthalmology, ECG parameters, urinalysis, ferritin, organ weights, or macroscopic and microscopic examinations.

[0273] Administration ofXPATOl in monkeys elicited changes in clinical pathology parameters at >0.15 mg / kg / dose. These changes were more pronounced following the first infusion and were either no longer observed on the subsequent infusion or of a lesserVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO magnitude. Hematology changes consisted of mild to moderate decreases in lymphocyte counts at >0.5 mg / kg / dose on Days 1 and 8, 24 hours post-start of infusion (SOI). Monocyte counts appeared minimally decreased at >0.1 mg / kg / dose. Coagulation changes consisted of minimal prolongation of prothrombin and activated partial thromboplastin times at 1.5 mg / kg / dose on Day 1, 24 hours post-SOI. Fibrinogen concentrations were increased at > 0.15 mg / kg / dose on Day 1, 24 hours post-SOI, and at >0.5 mg / kg / dose on Day 22, 24 hours post-SOI. Clinical chemistry changes included minimal to moderate increases in CRPs at >0.15 mg / kg / dose on Day 1, 24 hours post-SOI, and at >0.5 mg / kg / dose on Day 22, 24 hours post-SOI. Changes observed in coagulation parameters and in CRP were indicative of a transient acute-phase response and may have been, in part, procedure related. All changes previously mentioned were no longer observed 7 days after the dosing and at the end of the recovery period.

[0274] Transient XPAT01 -related cytokine changes were observed on Day 1 starting 7 hours post-SOI and up to 48 hours post-SOI. These changes included increases of IL-6 in females at >0.5 mg / kg / dose and MCP-1 at 1.5 mg / kg / dose.

[0275] For IL-6, no changes were observed in animals dosed at 0.15 mg / kg / dose or males dosed at 0.5 mg / kg / dose. For females dosed at 0.5 or 1.5 mg / kg / dose, increases in IL-6 ranging from 1.4x to 17.3x were observed mainly at 7 to 48 hours post-SOI on Day 1. IL-6 concentrations returned to baseline by Day 8, except in 2 females at 1.5 mg / kg / dose that exhibited increases of 6.2x to 17.3x on Day 22. For males dosed at 1.5 mg / kg / dose, postdose changes were observed in a single animal, which exhibited an increase of 2.9x at 1.5 hours post-SOI on Day 22.

[0276] Transient increases in MCP-1, ranging from 1.2x to 2.7 x, were observed in animals dosed at 0.15 or 0.5 mg / kg / dose, as well as in animals from the control group. For animals dosed at 1.5 mg / kg / dose, increases ranging from l.lx to 5. Ox were observed mainly on Day 1.

[0277] Overall, the changes observed in other cytokines evaluated (IFN-y, IL-2, IL- 10, IL-4, and TNF-a) were considered not XPAT01 -related.

[0278] Administration of XPAT01 resulted in changes in immunophenotyping parameters. XPAT01 induced a pattern of an initial decrease followed by a return to baseline or increase in the lymphocyte counts. An XPAT01 dose-related rapid decrease on Days 1, 8, and 15 inVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO total lymphocyte counts and absolute counts of all lymphocyte populations of interest was observed 24 hours post-SOI, followed by an increase above baseline 1 week after infusion at >0.5 mg / kg / dose and in some animals dosed at 0.15 mg / kg / dose. The greatest decrease was observed in the T lymphocyte subpopulations (total T, helper T and cytotoxic T lymphocytes), based on the fold change relative to the control group, leading to a transient imbalance between T cells, natural killer (NK) cells, and B cells. On Day 1, 24 hours postdose, a decrease in the relative percentage of total T cells ( T helper and T cytotoxic) and NK cells, with an increase in the relative percentage of B cells, was observed in males and females dosed at >0.5 mg / kg / dose and in some animals dosed at 0.15 mg / kg / dose. A similar pattern was observed on Days 8 and 15, 24 hours postdose, although the changes were not present in all animals and were overall of lower magnitude. At later timepoints, the magnitude of changes decreased, especially in the high-dose group. On Day 22, a non-dose-dependent decrease in the absolute counts of all cell populations of interest was still observed but only in some animals 24 hours postdose. During the recovery period, the absolute counts and relative percentages of T, B, and NK lymphocytes in dosed animals returned to baseline levels.

[0279] XPAT01 also induced the activation of helper T and cytotoxic T lymphocytes as observed by an increase in the expression of CD69. Twenty-four hours following dosing on Day 1, a dose-dependent increase in CD69 was observed at the surface of helper T cells at >0.5 mg / kg / dose and cytotoxic T cells at >0.15 mg / kg / dose. On Day 8, prior to dosing, the expression of CD69 returned to near baseline levels and increased again upon dosing in animals dosed at >0.5 mg / kg / dose. On Days 15 and 22, some increases in CD69 expression were observed, but these changes lacked dose dependence and were generally of lower magnitude than those observed on Days 1 and 8. During the recovery period on Days 36 and 50, no changes in the expression of CD69 were observed.

[0280] In conclusion, the administration of XPAT01 by QW IV infusion in monkeys at levels of 0.15, 0.5, and 1.5 mg / kg / dose resulted in transient and reversible changes in hematology, coagulation, clinical chemistry, cytokines (IL-6 and MCP-1), and immunophenotyping parameters. All these changes were considered nonadverse based on their low magnitude and severity. Based on these results, the NOAEL was considered to be 1.5 mg / kg / dose, with a Cm ax of 252 nM on Day 1.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Other toxicity studies

[0281] The effects of single-dose administration of metabolites of XPAT01 were investigated in non-GLP Studies AX22-01 [fully unmasked metabolite XPAT01-TCE] and AX22-03 [partially unmasked metabolites XPATOl(lx-N) and XPATOl(lx-C)].

[0282] Animals received a single administration of an aqueous solution of XPAT01-TCE in 0.9% sodium chloride for injection at 0.02, 0.06, or 0.15 mg / kg by a 30-minute IV infusion. The parameters evaluated included mortality, clinical observations, body temperature, body weight, qualitative food consumption, clinical pathology (hematology and clinical chemistry), and cytokine analysis. Blood samples for hematology and clinical chemistry were collected prior to dosing and 24 hours post-EOI. Histopathology was not performed.

[0283] A single administration of XPAT01-TCE at 0.02 and 0.06 mg / kg was well tolerated. XPATOl-TCE-related clinical pathology changes included a decrease in mean corpuscular volume and an increase in AS1', ALT, GRP, CK, and ferritin. A single administration of XPAT01-TCE at the 0.15-mg / kg dose level caused loss in appetite and diarrhea and required supplemental food for recovery. XPATOl-TCE-related changes at 0.15 mg / kg included a decrease in lymphocytes and increases in neutrophils and white blood cells, as well as increases in AST, ALT, BUN, GRP, ferritin, CK, and troponin.

[0284] A dose-dependent increase in all quantified cytokines (MCP-1, GM-CSF, IFN-y, IL-ip, IL-10, IL-2, IL-4, IL-6, and TNF-a) was observed after administration of XPAT01-TCE. This increase was highest with 0.15 mg / kg XPAT01-TCE. In general, peak cytokine concentrations were observed between 2 and 6 hours post-EOI, with levels decreasing by 12 hours post-EOI.For XPATOl(lx-N) and XPATOl(lx-C)]

[0285] Two male and 2 female monkeys received a single administration of 0.2 mg / kg XPATOl(lx-N) or 0.1 mg / kg XPATOl(lx-C). The duration of the IV infusion for each administration was 60 minutes.

[0286] The parameters evaluated included mortality, clinical signs, body temperature, body weight, qualitative food consumption, cytokines (IL-ip, IL-2, IL-4, IL-6, IL-10, IFN-y, TNF-a, MCP-1), hematology, clinical chemistry, coagulation, and histopathology.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0287] Single-dose administrations of XPATOl(lx-N) at 0.2 mg / kg or XPATOl(lx-C) at 0.1 mg / kg were well tolerated, with all animals surviving to scheduled necropsy on Day 8. After a single administration of 0.1 mg / kg of XPATOl(lx-C), the male animal had 11% BWL on Day 8 (necropsy). The male animal treated with 0.2 mg / kg XPATOl(lx-N) had a transient increase in body temperature 6 hours post-EOI, with a decreasing level observed 24 hours post-EOI.

[0288] The clinical chemistry changes induced by the partially masked metabolites of XPAT01 included CRP and ALT (without concurrent increase in AST) elevations in the animals treated with XPATOl(lx-N) and XPATOl(lx-C) on Day 1, 24 hours post-EOI.

[0289] Microscopic changes following single administration of XPATOl(lx-N) or XPATOl(lx-C) were limited to the lymphoid organs (spleen, gut-associated lymphoid tissue, and mandibular, mesenteric, and draining lymph nodes) and were characterized by minimal to mild increased lymphoid follicles, paracortical hyperplasia, and increased mitosis in the lymph nodes.Summary

[0290] The nonclinical safety profile of XPAT01 was evaluated in studies in monkeys, which were the only relevant toxicology species based on the binding profile. The definitive GLP repeat-dose study was 4 weeks in duration with a 4- week recovery period. Doses ranged from 0.15 to 1.5 mg / kg / week given by IV infusion. Safety pharmacology core battery studies as defined in ICH S7A and S7B guidelines were not conducted; instead, safety pharmacology endpoints were incorporated in the GLP repeat-dose study.

[0291] Dose-limiting toxi cities (DLTs) were seen at single doses >8 mg / kg. These were considered to be related to acute cytokine release based on clinical observations, clinical chemistry and hematology changes, and increases in cytokine levels. Acute cytokine release is consistent with the MOA of XPAT01, which is a T-cell-engaging compound. Attenuation of cytokine release was observed following repeat doses of XPAT01. XPAT01 -related changes identified during exploratory and definitive toxicity studies are consistent with the MOA of the drug.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Example 2. Clinical Evaluation of XPAT01

[0292] This is a Phase 1 , first-in -human study of the safety, pharmacokinetics, and preliminary efficacy of XPAT01 alone and in combination with pembrolizumab in participants with locally advanced or metastatic solid tumors.

[0293] XPAT01 is supplied as a single-use, sterile, nonpyrogcnic, white to off-white lyophilized powder for solution for intravenous (IV) infusion in glass vials. Once reconstituted with the appropriate amount of sterile, preservative-free WEI, EP / USP, each milliliter of XPAT01 contains 2 mg of XPAT01, citric acid anhydrous (0.39 mg), trisodium citrate anhydrous (4.64 mg), polysorbate 80 (PS-80) (0.2 mg), trehalose anhydrous (80 mg), and hydrochloric acid or sodium hydroxide to adjust pH to 6.3. After reconstitution with sterile, preservative-free WFI, EP / USP, the resulting concentration of XPAT01 is 2 mg / mL at a pH of 6.3.Overall design synopsis

[0294] This Phase 1, first-in-human (FIH), dose -escalation and dose-expansion study is designed to evaluate the safety, PK, and preliminary anti-tumor activity of XPAT01 as a monotherapy and in combination with pembrolizumab in participants with solid tumors that are known to express EGFR. The study will be conducted in the following 4 parts (FIG.15 and 16A-16C):• Part 1: XPAT01 monotherapy dose escalation• Part 2: XPAT01 monotherapy dose expansion• Part 3: XPAT01 plus pembrolizumab dose escalation• Part 4: XPAT01 plus pembrolizumab dose expansion

[0295] Parts 1 (XPAT01 monotherapy dose escalation) and 3 (XPAT01 plus pembrolizumab dose escalation) are designed to characterize the safety and PK profiles of XPAT01, as well as establish a recommended dose(s) for expansion cohorts. The XPAT01 recommended dose(s) for expansion cohorts will be evaluated in Parts 2 (XPAT01 monotherapy dose expansion) and 4 (XPAT01 plus pembrolizumab dose expansion), respectively, to explore the preliminary anti tumor activity and further characterize the safety profile of XPAT01.XPAT01 in combination with pembrolizumabVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0296] Part 3 of the study will focus on dose escalation of XPAT01 in combination with pembrolizumab. The safety assessment committee (SAC) will determine the starting dose of XPAT01 in Part 3 based on the totality of the data, which will be at least 1 dose level below the monotherapy maximum assessed dose that has cleared the DLT assessment period and has been deemed tolerable.XPAT01 Dosage

[0297] Part 1 of the study will evaluate escalating doses of XPAT01 as monotherapy administered as a fixed dose, QW. The goal of Part 1 is to determine the recommended dose(s) for expansion cohorts for the QW schedule. The starting dose will be 3 pg / kg. If de-escalation is warranted, the dose administered will be no less than 1 pg / kg.

[0298] Part 3 of the study will focus on dose escalation of XPAT01 QW in combination with pembrolizumab 200 mg Q3W. The starting dose of XPAT01 QW in combination with pembrolizumab will be at least 1 dose level below the monotherapy QW maximum assessed dose that has cleared the DLT assessment period.

[0299] The recommended starting dose of XPAT01 is 0.003 mg / kg (3 pg / kg) QW administered by IV infusion over 2 hours; this was selected based on the totality of data and using the most conservative approach after considering multiple methods for FIH starting dose selection, as summarized in Table 10.Table 10. Considerations for determination of the XPAT01 first-in-human starting dose FIH starting dose (mg / kg) MRSD1 / 10 NHPNOAEL (1.5 mg / kg in the GLP study) 0.15 MABELCombined activities of XPAT01 and its metabolites on normal humankeratinocytes 0.3 XPAT01 activity on MDA-MB-231 cells 0.01 Combined activities of XPAT01 and its metabolites on MDA-MB-231 0.003 cellsProposed FIH starting dose based on the totality of data 0.003Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Table 11. Safety margin of the first-in-human starting doseSpecies Cmax Dose Cynomolgus monkey 252 nMa1.5 mg / kgbHuman 0.457 nMc0.003 mg / kg Safety margin 551 500a Observed mean Day i Cmaxin the repeat-dose GLP toxicology study in cynomolgus monkeys at the NOAEL (1.5 mg / kg). Overall mean Cmax determined based on male and female Day 1 Cmaxvalues as presented in the IB.b NOAEL of the repeat-dose GLP toxicology study in cynomolgus monkeys (highest dose tested). c Predicted Cmaxof proposed first-in-human starting dose of 0.003 mg / kg (3 iig / kg ) IV for XPAT01 (based on a 60-kg patient with a volume of distribution of 2.8 L and calculated for an IV bolus administration). As XPAT01 will be administered by IV infusion over 2 hours, the actual Cmaxof the proposed starting dose will be slightly lower.Step-up dosing

[0300] Dose-escalation cohorts (Parts 1 and 3) may adopt step-up dosing of XPAT01. This approach will enable the evaluation of XPAT01 administered QW in escalating doses during Cycle 1, which is expected to further reduce the risk of CRS.

[0301] A step-up escalation dose cohort(s) may use either 1-step or 2-step intraparticipant dose escalation of XPAT01 in Cycle 1. For each new dose level of XPAT01 in Cycle 1 exceeding the previously administered dose level, participants may be hospitalized and monitored for at least 24 hours following dosing. The initial Cycle 1 Day 1 XPAT01 dose for evaluation should be less than or equal to a dose level that has previously been evaluated and cleared the DLT window.Dose-escalation rules

[0302] The BOIN (Bayesian optimal interval) design may be used to monitor DLTs during dose escalation and determine the MTD. The target DLT rate for the MTD is $ = 0.3. An accelerated titration option may be used (in Part 1 only) for the first cohorts, up to a dose of below 30 pg / kg (with n = 1 per cohort), unless any AE of Grade >2 (except for fatigue or toxicities clearly related to disease progression or intercurrent illness) or a DLT is observed.

[0303] Starting at level-s >30 pg / kg, the minimum cohort size for each dose level may be 3 participants. Dose level increments will be no more than approximately half-log increases. Dose increments for step-up and full treatment doses for the following and all future cohorts will be limited under the following conditions:Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO • 100% if participants of a given cohort experience 1 Grade 2 CRS lasting longer than 3 days;• 50% if participants experience more than 1 Grade 2 CRS lasting longer than 3 days;• 50% if 1 DLT occurs during the DLT period; and• 30% if 2 DLTs occur during the DLT period.

[0304] The BOIN design uses the following rules, optimized to minimize the probability of incorrect dose assignment, to guide dose escalation / de-escalation:• If the observed DLT rate at the current dose is <0.236, the dose may be escalated to the next-higher dose level;• If the observed DLT rate is >0.333, the dose should be de-escalated to the next-lower dose level;• If the observed DLT rate is >0.236 but <0.333, additional participants may be evaluated at the current dose;• If a cohort consists of 3 participants, a “3+3 run-in” rule will be applied, for which 1 DLT in 3 participants would result in staying at the current dose.

[0305] For the purpose of overdose control, doses j and higher levels will be eliminated from further consideration if Prob(pj >0.3 I data) >0.95 and at least 3 evaluable participants have been treated at dose level j, where pj is the true DLT rate of dose level j, j = 1 , ..., total number of dose levels. This posterior probability is evaluated based on the beta-binomial model yj I Pj ~ binomial(pj) with pj ~ uniform(0, 1), where yj is the number of participants who experienced a DLT at dose level j. When the lowest dose is eliminated, dose escalation will be stopped for safety. The probability cutoff of 0.95 has been chosen to be consistent with the common practice that when the target DLT rate ({) is <1 / 6, a dose with two -thirds of participants experiencing DLTs is eliminated.

[0306] The steps to implement the BOIN design are described as follows:Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Participants in the first cohort are treated at Dose Level 1. Note: if a participant in the first dosing cohort experiences Grade >3 CRS or neurotoxicity, dose de-escalation may be recommended;To assign a dose to the next cohort of participants, dose escalation / de-escalation parameters will be determined. After the accelerated titration option (up to a dose below the 30-pg / kg dose level) has ended, a minimum of 3 participants will be evaluated at higher dose levels. As per the BOIN design, the aim is to minimize participant exposure to a dose that exceeds a DLT rate of >0.333 but also allows the flexibility to evaluate additional participants to better establish the true DLT rate in an otherwise heterogeneous patient population. Decisions to enroll additional participants at the existing dose level, dose de-escalate, or dose escalate will be made based on all available data:If a DLT occurs in 1 of 3 participants, 3 additional participants will be enrolled in that cohort;If the BOIN model does not support the recommendation, dose escalation will not be recommended. Furthermore, based on all available data, the trial may choose not to follow a BOIN recommendation for dose escalation to protect participant safety. On the other hand, the following recommendation should be followed: 1) to dose dc-cscalatc or 2) for additional enrollment of participants in a cohort to evaluate a DLT rate;If none of the actions (i.e., dose escalation, de-escalation, or elimination) is triggered, new participants will be treated at the current dose level. If new participants are enrolled before all 3 initial participants have cleared the DLT assessment period and the final DLT rate exceeds 0.333, the trial may de-escalate the dose to the next-lower dose level;If the current dose is the lowest dose level and the rule indicates dose de-escalation, new participants may be treated at the lowest dose level, unless the number of DLTsVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO reaches the elimination boundary, at which point the study will be stopped for safety reasons;7. If the current dose is the highest planned dose level and the rule indicates dose escalation, new participants will be treated at the highest dose level;8. Repeat Step 2 until the maximum sample size of approximately 75 (Part 1) is reached or stop dose escalation early if the number of evaluable participants treated at the current dose level is >9.

[0307] Planned fixed and step-up dose levels are illustrated in Table 12. Alternative dose levels may be explored based on the emerging safety, PK, and activity data.Table 12. XPAT01 Dose Escalation: Fixed and Step-Up Dosing RegimensCycle 1 dose (pg / kg)Cycle 2 andDose level DI D8 D15 beyond (pg / kg)1 3 3 3 32 10 10 10 103 30 30 30 304 too 100 100 1005 too 200 200 2006 too 200 400 4007 200 400 800 8008+ TBD TBD TBD TBDAbbreviations: D, Day; TBD, to be determined.

[0308] The proposed starting dose of 0.003 mg / kg (3 pg / kg) QW was selected to minimize the potential for CRS and / or IRRs and is based on the most conservative of the maximum recommended starting dose (MRSD) and MABEL approaches. The MRSD dose was considered using a no-observed-adverse-effect level (NOAEL) approach. MABEL doses were considered using the cytotoxicity of XPAT01 or the combined cytotoxicity of XPAT01 and its metabolites in MDA-MB-231 cells, the most sensitive endpoint of the most relevant in vitro assay for XPAT01. The MABEL dose was also considered using the combined cytotoxicity of XPAT01 and its metabolites in normal human keratinocytes.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO

[0309] The recommended starting dose of 3 pg / kg XPAT01 is predicted to yield a plasma Cmax of 0.457 nM. This concentration is approximately 551 -fold lower than the observed Cmax of the NOAEL in the 1 -month repeat -dose GLP toxicity study in monkeys (Table 11).

[0310] The proposed QW dosing frequency of XPAT01 in the initial dose escalation of Part 1 is supported by the predicted human clearance and t1 / 2of XPAT01 and by the 4-week cynomolgus monkey GLP toxicology study with a 4-week recovery period, in which XPAT01 was generally well tolerated when administered QW for 4 weeks; no XPAT01 -related mortalities or significant changes or acute (or other) toxicities were observed. Alternative dosing schedules for XPAT01, including Q2W (FIG. 15B), Q3W (FIG. 15C), or other schedules may be explored based on emerging clinical safety, efficacy, and PK data and upon recommendation of the SAC. A Q2W or Q3W schedule would be less burdensome for patients and more conducive to coadministration with partner therapies such as pembrolizumab. The underlying assumption is that the recommended dose(s) for expansion cohorts for the Q2W or Q3W schedules would be approximately 2 or 3 times higher than the QW recommended dose(s) for expansion cohorts.

[0311] The planned dose of pembrolizumab for this study is 200 mg Q3W when used in combination with XPAT01 administered on a QW or a Q3W schedule. The planned dose of pembrolizumab for this study is 400 mg Q6W when used in combination with XPAT01 administered on a Q2W schedule. A 400-mg Q6W dosing regimen of pembrolizumab is expected to have a similar benefit-risk profile as that of 200 mg Q3W in all treatment settings in which 200 mg Q3W pembrolizumab is currently appropriate. Specifically, the pembrolizumab 400-mg Q6W dosing regimen is considered adequate based on modeling and simulation analyses.

[0312] The first XPAT01 Q2W dose level evaluated may not exceed an approximate half- log increase in projected Cmax or projected AUC from any maximum administered monotherapy dose that has cleared the DLT window. The Q2W dose level increment for XPAT01 will be determined based on the totality of the PK and safety data, and it may be no more than an approximately half- log increase from the prior Q2W dose level that has cleared the DLT window.

[0313] The trial may evaluate escalating doses of XPAT01 administered as a Q2W fixed dose on a 28-day cycle in combination with 400 mg pembrolizumab administered on a Q6WVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO schedule. The first XPAT01 Q2W dose in combination with pembrolizumab can be at least 1 dose level below the maximum administered XPAT01 Q2W monotherapy dose that has cleared the DLT window.

[0314] Cycle 1 may be 21 days in length and may consist of 1, 2, or 3 doses of XPAT01 administered QW or Q3W. In some embodiments, there may be one, two, or three step up doses of XPAT01 at the beginning of Cycle 1. Starting with Cycle 2 on Day 1, weekly administering of XPAT01 may be at the highest step up dose level, also referred to herein as a target dose (FIG. 16A). In some embodiments, Q3W dosing can be implemented at the highest step up dose (FIG. 16C). In some embodiments, XPAT01 is administered in combination with 200 mg pembrolizumab administered on a Q3W schedule.

[0315] Cycle 1 may be 28 days and may consist of 2, 3, or 4 doses of XPAT01 administered QW or 2 doses of XPAT administered Q2W. In some embodiments, there may be one, two or three step up doses of XAPT01 at the beginning of the Cycle 1. The fourth dose can be administered at the highest step up dose level (FIG. 16B). Starting with Cycle 2 on Day 1, Q2W dosing can be implemented at the highest step up dose, also referred to herein as a target dose, for all subsequent 28 day cycles. In some embodiments, the XPAT01 is administered in combination with 400 mg pembrolizumab administered on a Q6W schedule.

[0316] The first XPAT01 Q3W dose level evaluated may not exceed an approximate half- log increase in projected Cmax or projected AUC from any maximum administered monotherapy dose that has cleared the DLT window. The Q3W dose level increment for XPAT01 will be determined based on the totality of the PK and safety data, and it may be no more than an approximately half-log increase from the prior Q3W dose level that has cleared the DLT window.

[0317] The first XPAT01 Q3W dose in combination with pembrolizumab may be at least one dose level below the maximum administered XPAT01 Q3W monotherapy dose that has cleared the DLT window.Inclusion Criteria

[0318] Participants are eligible to be included in the study only if all of the criteria as shown in Table 13.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Table 13. Diseases under study, lines of therapy, and biomarker statusCohort CriteriaParts 1 and 3 (dose • Have one of the following:escalation) > NSCLC (non squamous or squamous histology), CRC, HNSCC, or CSCCNote: Participants with nasopharyngeal tumors are eligible.Note: Participants with upper esophageal or salivary gland tumors are not eligible.• Have a solid tumor with EGFR amplification (as previously determined locally with an analytically validated assay in a certified testing laboratory)• Have no available standard systemic therapy; or standard therapy is intolerable, not effective, or not accessible; or participant has refused standard therapyCohorts 2A and 4A • Received (or ineligible for) both irinotecan- and oxaliplatin-based (CRC) chemotherapy (± bevacizumab per local practice, ± anti-EGFR antibody [RAS wild-type only] per local practice, unless contraindicated)• Have received no more than 4 prior therapeutic regimens for metastatic disease• Note: Participants with known MSI-H or dMMR status are not eligible.Testing for MSI-H or dMMR is per local test.Note: Participants with known BRAF activating mutation are not eligible. Testing for BRAF is per local test.• Note: Enrollment of participants with RAS mutation will be capped al 50% of total cohort enrollment. Testing for RAS status is per local test. Cohorts 2B and 4B • Received platinum-based chemotherapy and anti-PD-(L)l therapy (NSCLC withsquamous histology)Cohorts 2C and 4C • Received an anti-EGFR therapy (per local approved label, if applicable) (NSCLC with EGFR-eReceived platinum-based chemotherapy [± bevacizumab and ± anti-PD- activating mutation) (L)l therapy per local practice] unless ineligible or intolerant Cohorts 2D and 4D • Received an an ti-PD-(L)l therapy(HNSCC)eReceived (or ineligible for) platinum-based chemotherapyNote: Participants with nasopharyngeal tumors are eligible.Note: Participants with upper esophageal or salivary gland tumors are not eligible.Note: Participants with oropharyngeal tumors with HPV-positive status are not eligible. Testing for HPV status is per local test.Cohorts 2E and 4E Have a solid tumor with EGFR gene amplification (as previously (solid tumor with determined locally with an analytically validated assay in a certified EGFR amplification) testing laboratory)Have no available standard systemic therapy available for the participant’s tumor histology and / or molecular biomarker profile; or standard therapy is intolerable, not effective, or not accessible; or participant has refused standard therapyAbbreviations: CRC, colorectal cancer; CSCC, cutaneous squamous cell carcinoma; dMMR, deficient mismatch repair; EGFR, epidermal growth factor receptor; HNSCC, head and neck squamous cell carcinoma; HPV, human papilloma virus; MSI-H, microsatellite instability-high; NSCLC, non -small-cell lung cancer; PD-(L)1, programmed cell death (ligand) 1; RAS, rat sarcoma.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO Exclusion Criteria

[0319] Participants are excluded from the study if any of the following criteria (Table 14).Table 14. Exclusion CriteriaExclusion category CriteriaPregnancy exclusion — Are a WOCBP with a positive serum or urine pregnancy test within72 hours prior to treatment.Medical conditions — Have acute or chronic infections, including the following:• Acute or chronic active Epstein-Barr virus (EBV) infection (Exception:asymptomatic EBV-positive participants are still eligible)• Chronic active EBV disease defined as a chronic illness lasting at least 6 months, an increased EBV level in either the tissue or the blood, and lack of evidence of a known underlying immunodeficiency• History of hepatitis B infection (defined as hepatitis B surface antigen [HBsAg] reactive) or known active hepatitis C virus (HCV) infection (defined as HCV [HCV RNA; qualitative] is detected)• History of HIV infection. No HIV testing is required unless mandated by the local health authority• Active infection requiring systemic therapy within 14 days of Cycle 1 Day 1• Known positive COVID-19 test result at screening (Exception: If follow-up test is negative, participants may be eligible if asymptomatic and upon consultation with medical monitor)— Have a concomitant medical or inflammatory condition that may increase the risk of toxicity to XPAT01 or pembrolizumab— Have a QT interval corrected by Fridericia’s method (QTcF) that is >480 msPrior / concomitant - Have received prior systemic anti-cancer therapy, including investigational therapy and adverse agents, within 5 half-lives prior to first dose of study intervention. For drugs events with a long ti / z, such as mAbs, or for drugs for which the ti / 2 is not known, the last dose should not have been within 28 days prior to first dose of study intervention.— Have received prior radiotherapy within 2 weeks of start of study intervention— Have had a prior Grade >3 irAE, such as pneumonitis, colitis, hepatitis, or nephritis. Prior dermatitis and endocrinopathies are allowed, provided corticosteroids are no longer required and endocrine replacement therapy is stable (replacement with chronic corticosteroids is allowed)- Have received prior therapy with an anti-PD-1, anti-PD-Ll, or anti-PD-L2 agent or with an agent directed to another stimulatory or coinhibitory TCR (e.g. , CTEA-4, 0X40, CD! 37), AND was discontinued from that treatment due to a Grade 3 or higher irAE.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO — Have received a live or live-attenuated vaccine within 30 days prior to the first dose of study intervention.— Have had an allogeneic tissue / solid organ transplant.Prior / concurrent - Are currently participating in or have participated in a study of an clinical study investigational agent within 5 half-lives prior to the first dose of study experience intervention. For drugs with a long ti / 2, such as mAbs, or for drugs for which the ti / 2 is not known, the last dose should not have been within 28 days prior to first dose of study intervention.Medical history- Have a diagnosis of immunodeficiency or are receiving chronic supraphysiological systemic steroid therapy (dosing exceeding 10 mg daily of prednisone equivalent) or any other form of immunosuppressive therapy within 7 days prior the first dose of study intervention— Have a known additional malignancy that is progressing or have required active treatment within the past 3 years— Note: Participants with untreated in situ basal cell carcinoma, or with basal cell carcinoma of the skin, squamous cell carcinoma of the skin, transitional cell carcinoma or urothelial cancer, or carcinoma in situ (excluding carcinoma in situ of the bladder) who have undergone potentially curative therapy and participants with low-risk papillary thyroid carcinoma (at the investigator’s discretion) are not excluded.- Have known active CNS metastases and / or carcinomatous meningitis.Participants with previously treated brain metastases may participate, provided they are radiologically stable, i.e., without evidence of progression for >4 weeks by repeat imaging (performed during study screening), are clinically stable, and do not require steroid treatment for >14 days prior to first dose of study intervention- Have had severe hypersensitivity (Grade >3) to pembrolizumab (Parts 3 and 4 only) and / or any of its excipients- Have had severe hypersensitivity (Grade >3) to any XPAT01 excipients — Have currently active or history of any autoimmune disease (e.g. , rheumatoid arthritis) that has required or requires systemic treatment in the past 2 years (i.e., with the use of disease-modifying agents, corticosteroids, or immunosuppressive drugs). Replacement therapy (e.g., thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered a form of systemic treatment and is allowed.— Have a history of (noninfectious) pneumonitis / interstitial lung disease (ILD) that required steroids or have current pneumonitis / ILD— Have an arterial oxygen saturation (SaOz) <92% (at rest, on room air) at baseline- Have ulcerative colitis, Crohn’s disease, or other inflammatory intestinalconditions requiring treatmentVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO- Have unresolved corneal disorders or any previous corneal disorders that are considered by an ophthalmologist to place the participant at higher risk of drug-induced keratopathy- Have a known psychiatric or substance abuse disorder that, in the opinion of the investigator, would interfere with the participant’s ability to cooperate with the requirements of the study- The following applies for NSCLC participants:• Single-agent dose escalation and dose expansion (Parts 1 and 2): Have received radiation therapy to the lung that is >30 Gy within 3 months of the first dose of trial treatment• Pembrolizumab combination dose escalation and dose expansion (Parts 3 and 4): Have received radiation therapy to the lung that is >30 Gy within 6 months of the first dose of trial treatmentOther exclusioncriteria — Have lesions in proximity of vital organs that, in the opinion of the investigator, may lead to sudden decompensation or deterioration in the setting of a tumor flare, including, but not limited to, untreated spinal lesions at risk of causing cord compression or intestinal lesions that may lead to perforation or obstruction— Have uncontrolled tumor-related pain— Have a history of severe hypersensitivity reactions to mAbs or other therapeutic proteins— Are accommodated in an institution because of regulatory or legal order — prisoners or participants who are legally institutionalized— Are not suitable for participation, whatever the reason, as judged by the investigator, including for medical or clinical conditions, or are potentially at risk of noncompliance to study procedures— Are employees of the clinical study site or other individuals directly involved in the conduct of the study, or immediate family members of such individuals (in conjunction with Section 1.61 of International Council for Harmonisation [ICH] Good Clinical Practice [GCP] Ordinance E6)— Have a sensitivity to any of the study interventions, or components thereof, or drug or other allergy that, in the opinion of the investigator, contraindicates participation in the study— Fall under any country-related specific regulation that would prevent the participant from entering the study.Premedication

[0320] Premedication can be administered prior to dosing of study treatment during Cycles 1 through 3 unless contraindicated. Cases in which a participant has a medical condition that precludes pretreatment should be discussed with the medical monitor. After Cycle 3, the useVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO of premedication is encouraged. The premedication regimen should be given 30 to 60 minutes before the start of XPAT01 or pembrolizumab infusion (if applicable), and can include the following: antihistamine (an oral or IV Hl receptor antagonist such as 50 to 100 mg diphenhydramine (or equivalent, including promethazine)), or antipyretic (an oral or IV antipyretic agent such as 325 to 1000 mg acetaminophen (or equivalent)).

[0321] The specific drugs and doses can be selected according to the institutional guidelines. Although continued premedication is recommended for all XPAT01 administrations, after Cycle 3, the trial may adjust the dosage and / or avoid using certain prcmcdication agents during the treatment, if supported by assessment of the risk from CRS or other IRRs.

[0322] If indicated after Cycle 1 Day 1, IV corticosteroids such as 20 mg dexamethasone IV or equivalent for IRRs and 2 mg / kg / day methylprednisolone or equivalent or pretreatment with IV fluids for CRS may be administered as premedication prior to XPAT01 administration.* * *

[0323] The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

[0324] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO CLAIMSWhat is claimed is:

1. A method for treating cancer in a patient in need thereof, comprising administering to the patient a dose of at least 1 pg / kg of a polypeptide once every week to once every eight weeks, wherein the polypeptide comprises (a) a first extended recombinant polypeptide (XTEN) having at least 90% sequence identity to SEQ ID NO:2, (b) a core fragment comprising an anti-EGFR antigen-binding fragment and an anti-CD3 antigen-binding fragment, and (c) a second XTEN having at least 90% sequence identity to SEQ ID NO: 12, and wherein (a) is connected to (b) through a first protease-cleavable site, and (b) is connected to (c) through a second protease-cleavable site.

2. A method for enhancing immune cell infiltration into a solid tumor in a cancer patient, comprising administering to the patient a dose of at least 1 pg / kg of a polypeptide once every week to once every eight weeks, wherein the polypeptide comprises (a) a first extended recombinant polypeptide (XTEN) having at least 90% sequence identity to SEQ ID NO:2, (b) a core fragment comprising an anti-EGFR antigen-binding fragment and an anti-CD3 antigen-binding fragment, and (c) a second XTEN having at least 90% sequence identity to SEQ ID NO: 12, and wherein (a) is connected to (b) through a first protease-cleavable site, and (b) is connected to (c) through a second protease-cleavable site.

3. A method for treating cancer in a patient in need thereof, comprising administering to the patient a dose at least 1 pg / kg of a polypeptide once every week to once every eight weeks, wherein the patient is being treated with an anti-PD-1 or anti-PD-Ll inhibitor, wherein the polypeptide comprises (a) a first extended recombinant polypeptide (XTEN) having at least 90% sequence identity to SEQ ID NO:2, (b) a core fragment comprising an anti-EGFR antigen-binding fragment and an anti-CD3 antigen-binding fragment, and (c) a second XTEN having at least 90% sequence identity to SEQ ID NO: 12, and wherein (a) isVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO connected to (b) through a first protease-cleavable site, and (b) is connected to (c) through a second protease-cleavable site.

4. A method for enhancing immune cell infiltration into a solid tumor in a cancer patient, comprising administering to the patient a dose of at least 1 ug / kg of a polypeptide once every week to once every four weeks, wherein the patient is being treated with an anti-PD-1 or anti-PD-Ll inhibitor, wherein the polypeptide comprises (a) a first extended recombinant polypeptide (XTEN) having at least 90% sequence identity to SEQ ID NO:2, (b) a core fragment comprising an anti-EGFR antigen-binding fragment and an anti-CD3 antigen-binding fragment, and (c) a second XTEN having at least 90% sequence identity to SEQ ID NO: 12, and wherein (a) is connected to (b) through a first protcasc-clcavablc site, and (b) is connected to (c) through a second protease-cleavable site.

5. The method of claim 3 or 4, wherein the anti-PD-1 or anti-PD-Ll inhibitor is an anti-PD-1 or anti-PD-Ll antibody.

6. The method of claim 3 or 4, wherein the anti-PD-1 or anti-PD-Ll inhibitor is pembrolizumab.

7. The method of claim 6, wherein the pembrolizumab is administered at 200 mg once every three weeks, or 400 mg once every six weeks.

8. The method of any one of claims 3-7, wherein the cancer is characterized with PD-L1 expression.

9. The method of any one of claims 1-8, wherein the patient is administered 3 pg / kg to 800 pg / kg of the polypeptide once every week to once every four weeks.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO 10. The method of claim 9, wherein the patient is administered 30 pg / kg to 500 pg / kg of the polypeptide once every week to once every four weeks.

11. The method of any one of claims 1-10, wherein each dose of the polypeptide is between 0.2 mg and 200 mg.

12. The method of claim 11, wherein each dose is 0.2 mg, 0.5 mg, 0.75 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg.

13. The method of any one of claims 1-12, wherein the administration results in increase of GM-CSF, IFN-y, IL-10, IL-2, IL-4, TL-6, IL-10, MCP-1, or TNF-a.

14. The method of any one of claims 1-13, wherein the cancer is characterized with EGFR expression.

15. The method of claim 14, wherein the cancer is characterized with EGFR immunohistochemistry (1HC) grade 2+ or 3+, in situ hybridization positive (ISH+), or an activating EGFR mutation.

16. The method of claim 14, wherein the cancer is selected from the group consisting of breast cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, ovarian cancer, bladder cancer, colorectal cancer, endometrial cancer, head and neck cancer, lung cancer and salivary gland cancer.

17. The method of claim 14, wherein the cancer is colorectal cancer (CRC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinomas (HNSCC), cutaneousVir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO squamous cell carcinoma (CSCC), pancreatic ductal adenocarcinoma (PDAC) and renal cell carcinoma (RCC).

18. The method of claim 17, wherein the cancer patient has metastatic non-small cell lung cancer (NSCLC) or metastatic colorectal cancer (CRC) and has progressed on a prior treatment.

19. The method of claim 18, wherein the CRC is characterized as microsatellite stable (MSS).

20. The method of any one of claims 16-19, wherein the cancer patient is resistant to or has progressed from one or more lines of prior treatments.

21. The method of claim 20, wherein the one or more lines of prior treatments comprise an anti-EGFR therapy.

22. The method of claim 21 , wherein the anti-EGFR therapy comprises one or more of gefitinib, erlotinib, afatinib, brigatinib, icotinib, cetuximab, osimertinib, panitumumab, zalutumumab, nimotuzumab, and matuzumab.

23. The method of any one of claims 1-22, further comprising administering to the patient a steroid.

24. The method of claim 23, wherein the steroid is administered prior to the first administration of the polypeptide.

25. The method of claim 23, wherein the steroid is administered after detection of a cytokine release syndrome (CRS) or pneumonitis in the patient.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO26. The method of claim 25, wherein detection of the CRS comprises detection of an increase of IL-6 expression in the patient.

27. The method of any one of claims 23-26, wherein the steroid is dexamethasone, and the dexamethasone is administered for no more than 20 mg every 6 hours or longer.

28. The method of any one of claims 1-22, wherein the patient is not treated with a steroid during administration of the polypeptide or is not pre-treated with a steroid.

29. The method of any preceding claim, wherein the anti-EGFR antigen-binding fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:6 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NOG.

30. The method of any preceding claim, wherein the anti-CD3 antigen-binding fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:7.

31. The method of any preceding claim, wherein the first and second protease-cleavable sites each, independently, has at least 90% sequence identity to SEQ ID NOG.

32. The method of any preceding claim, wherein the polypeptide has at least 90% sequence identity to SEQ ID NO: 1.

33. The method of claim 32, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO34. A method for treating a solid tumor in a patient in need thereof, comprising administering to the patient a dose of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 once every one, two, three, four, five, or six weeks.

35. The method of claim 34, further comprising administering to the patient a first step-up dose of the polypeptide before the dose, wherein the first step-up dose is less than the dose.

36. The method of claim 35, further comprising administering to the patient a second step-up dose of the polypeptide after the first step-up dose and before the dose, wherein the second step-up dose is less than the dose and greater than the first step-up dose.

37. The method of claim 36, further comprising administering to the patient a third step-up dose of the polypeptide after the second step-up dose and before the dose, wherein the third step-up dose is less than the dose and greater than the second step-up dose.

38. The method of any one of claims 34-37, wherein the dose is between 0.2 mg and 200 mg.

39. The method of claim 38, wherein the dose is 0.2 mg, 0.5 mg, 0.75 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg.

40. The method of any one of claims 34-39, wherein the polypeptide is administered to the patient intravenously or subcutaneously.

41. The method of any one of claims 34-40, further comprising administering to the patient an additional therapeutic agent.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO42. The method of claim 41, wherein the additional therapeutic agent in administered to the patient intravenously or subcutaneously.

43. The method of claim 41 or 42, wherein the additional therapeutic agent is an immune checkpoint inhibitor, a tyrosine kinase inhibitor (TKI), a VEGF / VEGFR inhibitor, a RAS pathway inhibitor, a MEI' inhibitor, a PARP inhibitor, a CDK4 / 6 inhibitor, a bispecific antibody, a chemotherapy, or a radiotherapy.

44. The method of claim 43, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

45. The method of claim 44, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

46. The method of claim 45, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, and tislelizumab.

47. The method of claim 45, wherein the anti-PD-1 antibody is pembrolizumab.

48. The method of claim 47, wherein the method further comprises administering to the patient: (i) 200 mg of pembrolizumab once every three weeks, or (ii) 400 mg of pembrolizumab once every six weeks.

49. The method of claim 47 or 48, wherein the method comprises administering to the patient: (i) a dose of the polypeptide once every week, and (ii) 200 mg of pembrolizumab once every three weeks.Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO 50. The method of claim 47 or 48, wherein the method comprises administering to the patient: (i) a dose of the polypeptide once every two weeks, and (ii) 400 mg of pembrolizumab once every six weeks.

51. The method of claim 47 or 48, wherein the method comprises administering to the patient: (i) a dose of the polypeptide once every three weeks, and (ii) 200 mg of pembrolizumab once every three weeks.

52. The method of any one of claims 34-51, further comprising the patient receiving a pre-medication prior to the polypeptide.

53. The method of claim 52, wherein the pre-medication is selected from the group consisting of an antihistamine, an antipyretic, a steroid, an IL-6 receptor antagonist, and any combination thereof.

54. The method of any one of claims 34-53, wherein the solid tumor expresses epidermal growth factor receptor (EGFR).

55. The method of any one of claims 34-54, wherein the solid tumor is a lung cancer, prostate cancer, breast cancer, colorectal cancer (CRC), head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, pancreatic cancer, or skin cancer.

56. The method of claim 55, wherein the lung cancer is non-small cell lung cancer (NSCLC).

57. The method of claim 55, wherein the head and neck cancer is head and neck squamous cell carcinomas (HNSCC).Vir Ref. No.: S0240.W01PCT Attorney Docket No: 95KG-402128-WO58. The method of claim 55, wherein the skin cancer is cutaneous squamous cell carcinoma (CSCC).

59. The method of any one of claims 1-58, wherein the solid tumor is a metastatic solid tumor.

60. The method of any one of claims 1-59, wherein the solid tumor is characterized by EGFR gene amplification.

61. The method of any one of claims 1-60, wherein the patient has received a prior cancer therapy.

62. The method of claim 61, wherein the patient is refractory to the prior cancer therapy.

63. The method of claim 61 or 62, wherein the prior cancer therapy is an EGFR-directed therapy.

64. The method of claim 63, wherein the prior administered EGFR-directed therapy is a tyrosine kinase inhibitor (TKI) or an anti-EGFR antibody.

65. The method of any one of claims 1-64, wherein the patient expresses a biomarker predictive of a response to the polypeptide.

Images