Therapeutic uses of her-2 targeted bispecific polypeptides

Abstract

Provided are compositions and methods for treating cancers, in particular those characterized with HER2 expression. The compositions include a conditionally-activatable XTENylated Protease-Activated bispecific T Cell Engager targeting HER2 (HER2-XPAT) that remain inactive until activated by proteases present in tumor microenvironment. The HER2-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.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WOTHERAPEUTIC USES OF HER- 2 TARGETED BISPECIFIC POLYPEPTIDESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the United States Provisional Application Serial Nos. 63 / 737,533, filed December 20, 2024, and 63 / 829,578, filed June 24, 2025, the content of which is hereby incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (4O1153.xml; Size: (17,781 bytes; and Date of Creation: December 19, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] 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 of T cell activation that enhance and amplify the anti-tumor immune response. Thus, TCEs offer the promise of immunotherapy to patients whose tumors harbor insufficient mutations or have escaped immune surveillance by other means.

[0004] 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 the Ichnos ISB 1302 HER2 TCE at < 1 pg / kg doses highlights the challenge faced by TCEs even when directed against relatively tissue-restricted targets.

[0005] Clinical trials with blinatumomab, (an approved CD3xCD1 bispecific antibody) revealed that cytokine release syndrome (CRS) is one of the major safety-related adverse events. CRS and on-target toxicities at low drug doses have significantly compromised theVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO therapeutic index and potential of the TCE modality in the clinic against solid tumors. For example, the clinical trial for catumaxomab (CD3xEpCAM) was terminated due to drug- related hepatic failure at a lOpg dose. In another trial, a HER2-targeted TCE (Glenmark GBR1302) the dose of the agent was limited to less than Ipg / kg due to onset of G4 CRS. While pasotuxizumab (a PSMA-targeted TCE) showed a good response, it was hampered by CRS at doses greater than 40 pg / day. The literature is replete with other examples of the CRS and on-target toxicity challenges presented by TCEs.

[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 HER2 expression. The compositions include a conditionally-activatable XTENylated Protease- Activated bispecific T Cell Engager targeting HER2 (HER2-XPAT) that remain inactive until activated by proteases present in tumor microenvironment. The example HER2-XPAT, referred to as XPAT01, exhibited exceptional safety profiles even at doses at 1000 pg / kg. In particular, one of the main safety concerns commonly associated with T-cell engagers, cytokine release syndrome (CRS), were only observed at low grades so that no prophylactic use of steroids is necessary. Further, the excellent pharmacokinetic and pharmacodynamic profiles of the HER2-XPAT allow it to be used once per week or even less frequently.

[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 at least 100 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-HER2 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)Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-40H53-WO through a first protease-cleavable site, and (b) is connected to (c) through a second protease- cleavable site.

[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 at least 100 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-HER2 antigenbinding 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] In some embodiments, the patient is administered 300 pg / kg to 800 pg / kg of the polypeptide once every week to once every four weeks, optionally once every three weeks. In some embodiments, the patient is administered 400 pg / kg to 600 pg / kg of the polypeptide once every week to once every four weeks, optionally once every three weeks.

[0011] Still in another embodiment, provided is a method for treating cancer in a patient in need thereof, comprising administering to the patient at least 30 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-HER2 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.

[0012] Also provide dis a method for enhancing immune cell infiltration into a solid tumor in a cancer patient, comprising administering to the patient at least 30 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-HER2 antigen-binding fragment and an anti-CD3 antigen-binding fragment, and (c) a second XTEN having at least 90% sequence identity toVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOSEQ 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.

[0013] In some embodiments, the anti-PD-1 or anti-PD-Ll inhibitor is an anti-PD-1 or anti- PD-L1 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. In some embodiments, the cancer is characterized with PD-L 1 expression.

[0014] In some embodiments, the patient is administered 40 pg / kg to 500 pg / kg of the polypeptide once every week to once every four weeks, optionally once every three weeks. In some embodiments, the patient is administered 50 pg / kg to 70 pg / kg of the polypeptide once every week to once every four weeks, optionally once every three weeks.

[0015] In some embodiments, the polypeptide is administered to the patient at a first dose, a second dose later than and at least 40% higher than the first dose, and a third dose later than and at least 40% higher than the second dose. In some embodiments, the second dose is at least 80% higher than the first dose and the third dose is at least 80% higher than the second dose. In some embodiments, the third dose is 2 to 12 weeks after the first dose.

[0016] In some embodiments, the third dose is 400 pg / kg to 1500 pg / kg. In some embodiments, the third dose is 600 pg / kg to 1200 pg / kg.

[0017] In some embodiments, the third dose is 50 pg / kg to 600 pg / kg. In some embodiments, the third dose is 70 pg / kg to 400 pg / kg.

[0018] In some embodiments, the administration results in decrease of expression of CEA (carcinoembryonic antigen), CA9-19, ctDNA, or CA-125 in the patient.

[0019] In some embodiments, the cancer is characterized with HER2 expression. In some embodiments, the cancer is characterized with HER2 immunohistochemistry (IHC) grade 2+ or 3+, in situ hybridization positive (ISH+), or an activating HER2 mutation.

[0020] 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.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0021] 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-HER2 therapy. In some embodiments, the anti-HER2 therapy comprises one or more of trastuzumab, trastuzumab deruxtecan, tucatinib or lapatinib. In some embodiments, the cancer patient is a breast cancer patient that has undergone or is resistant to at least one line of prior treatments.

[0022] In some embodiments, the cancer patient has gastric cancer or gastroesophageal junction cancer and has undergone or is resistant to at least one line of prior treatments. In 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).

[0023] 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.

[0024] In some embodiments, the patient is not treated with a steroid during administration of the polypeptide or is not pre-treated with a steroid.

[0025] In some embodiments, the anti-HER2 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. 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 NO:3. In some embodiments, the polypeptide has at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0026] Also provided, in one embodiment, is a method for treating cancer in a patient in need thereof, comprising administering to the patient a polypeptide in a dosing regimen comprising: a first dosing cycle (Cl) comprising administering to the patient a target dose of the polypeptide; and a second dosing cycle (C2) comprising administering to the patient the target dose of the polypeptide; wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. It is contemplated that there can be an unlimited number of cycles.

[0027] In some embodiments, Cl comprises administering to the patient a first step-up dose of the polypeptide, a second step-up dose of the polypeptide, a target dose of the polypeptide, and optionally a third step-up dose of the polypeptide. In some embodiments, Cl comprises administering to the patient a first step-up dose of the polypeptide, a second step-up dose of the polypeptide, optionally a third step-up dose of the polypeptide, and a target dose of the polypeptide.

[0028] In some embodiments, the second step-up dose is administered to the patient prior to the target dose, and the first step-up dose is administered to the patient prior to the second step-up dose, and wherein the optional third step-up dose is administered to the patient prior to the target dose and after the second step-up dose.

[0029] In some embodiments, the first step-up dose is administered to the patient in week 1 of Cl, the second step-up dose is administered to the patient 6-8 days after the first step-up dose, and the target dose is administered to the patient 6-8 days after the second step-up dose.

[0030] In some embodiments, the first step-up dose is administered to the patient on Day 1 of Cl, the second step-up dose is administered to the patient on Day 8 of Cl, and the target dose is administered to the patient on Day 15 of Cl.

[0031] In some embodiments, the first step-up dose is administered to the patient in week 1 of Cl, the second step-up dose is administered to the patient 6-8 days after the first step-up dose, the third step-up dose is administered to the patient 6-8 days after the second step-up dose, and the target dose is administered to the patient 6-8 days after the third step-up dose.

[0032] In some embodiments, the first step-up dose is administered to the patient on Day 1 ofCl, the second step-up dose is administered to the patient on Day 8 of Cl, the third step-upVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO dose is administered to the patient on Day 15 of Cl, and the target dose is administered to the patient on Day 22 of Cl.

[0033] In some embodiments, C2 consists of administering to the patient a single target dose to the patient. In some embodiments, the target dose is administered to the patient on Day 1 of C2. In some embodiments, the target dose is between 300 mg to 1000 mg. In some embodiments, the target dose is 200 ug / kg, 300 ug / kg, 400 ug / kg, 500 ug / kg, 600 ug / kg, 700 ug / kg, 800 ug / kg, 900 ug / kg or 1000 ug / kg.

[0034] In some embodiments, the second step-up dose is less than the target dose. In some embodiments, the first step-up dose is less than the second step-up dose. In some embodiments, the third step-up dose is less than the target dose and greater than the second step-up dose. In some embodiments, the dosing regimen further comprises one, two, three, four, five, six, seven, eight, nine, or ten additional dosing cycles.

[0035] In some embodiments, the polypeptide is administered to the patient as a monotherapy. In some embodiments, the dosing regimen further comprises administering to the patient an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anti-PD-1 or anti-PD-Ll inhibitor. In some embodiments, the anti-PD-1 or anti- PD-L1 inhibitor is pembrolizumab. In some embodiments, the pembrolizumab is administered at 200 mg once every three weeks, or 400 mg once every six weeks.

[0036] In some embodiments, the cancer is characterized with HER2 expression. In some embodiments, the cancer is characterized with HER2 immunohistochemistry (IHC) grade 2+ or 3+, in situ hybridization positive (ISH+), or an activating HER2 mutation. In some embodiments, the cancer is selected from the group consisting of breast cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, ovarian cancer, urothelial cancer, bladder cancer, colorectal cancer, endometrial cancer, head and neck cancer, lung cancer and salivary gland cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 shows the results of in vivo evaluation of XPAT01, XPAT01-P1, XPAT01- TCE, and XPATOl-NoClvSite in the treatment of BT474 tumors in NOG mice engrafted with hPBMC. Abbreviations: QW, once a week; hPBMC, human peripheral blood mononuclear cell.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO

[0038] FIG. 2A-B show XPATOl’s pharmacokinetics during step-up dosing (A) and after a first dose (B) at different dose levels.

[0039] FIG. 3 shows tumor shrinkage in HER2+solid tumors.

[0040] FIG. 4 shows that XPAT01 was efficacious in heavily pretreated HER2+ CRC patients, in particular at doses ^400 pg / kg.

[0041] FIG. 5 shows that higher doses led to more rapid decline of tumor mass.

[0042] FIG. 6A-B show rapid and sustained decrease in CEA with deeper tumor shrinkage when escalated to higher doses in a HER2+CRC patient.

[0043] FIG. 7 shows that IL-6 level increases, a cytokine elevated during CRS, following XPAT01 administration were limited and transient.

[0044] FIG. 8 demonstrates the synergistic effects, in a BT-474 model, between XPAT01 and pembrolizumab. The combination anti-tumor effect of 99% was statistically significant (p < 0.0001) compared to XPAT01 (74%) or pembrolizumab (30%) alone.

[0045] FIG. 9A-B illustrate some example step-up dosing schedule for mono and combination therapies for XPAT01 .DETAILED DESCRIPTIONDefinitions

[0046] 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.

[0047] 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 termVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO“polypeptide” may be used instead of, or interchangeably with any of these terms. The term “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.

[0048] “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.

[0049] 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.

[0050] 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.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO retains the activity (e.g., epitope-binding) or structure (e.g., salt-bridge) of the reference sequence.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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, IgG2, IgGs, IgG4, IgGs, etc. are well characterized and are known to confer functionalVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-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.

[0055] 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)).

[0056] 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.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOThis 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.

[0057] 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).

[0058] 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.”

[0059] 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.: S0233.WO1PCTAttorney Docket No: 95KG-401153-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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.: S0233.WO1PCTAttorney Docket No: 95KG-401153-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.

[0064] 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.

[0065] 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.

[0066] 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 HER-2 Targeted Bispecific XPAT

[0067] 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.: S0233.WO1PCTAttorney Docket No: 95KG-401153-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.

[0068] 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 healthy tissue to T cell activation by TCEs results in an overall lack therapeutic index for these agents.

[0069] The present disclosure has tested a conditionally-activated TCE, XTENylated Protease- ctivated bispecific T Cell Engager targeting HER2 (referred to herein as HER2- 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.

[0070] The active core of XPAT01 includes two single chain antibody fragments (scFvs) targeting CD3 and HER2, respectively. Two unstructured polypeptide masks (XTENs) are attached to the core that sterically reduce target engagement of HER2 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.

[0071] 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 molecules, thus expanding the therapeutic index compared to unmasked TCEs.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO

[0072] 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.

[0073] 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-HER2 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 eight different proteases among three different classes over-expressed or dysregulated in cancer; these include several matrix metalloproteinases (MMPs), Matriptase, uPA, and the cysteine protease, legumain. As a safety checkpoint, co-engagement of both CD3 and HER2 by XPAT01 is required for T cell activation. Activation of T cells should not occur if XPAT01 is unmasked in inflamed tissues where HER2 expression is absent or if it encounters HER2 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 HER2 expression are present.

[0074] The presence of the XTEN on XPAT01 produces an prodrug 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.

[0075] 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. This translated to a safetyVir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO margin of greater than 400- fold. Presented differently, the XTEN masks of XPAT01 allowed for a 88-fold higher molar dose / kg of the XPAT01 prodrug to be administered than the fully activated XPAT01-TCE.

[0076] Also remarkably, in the Phase 1 human trial (Examples 2 and 3), maintenance doses of up to 800 pg / kg QW were tested and were shown to be tolerable and no maximum tolerated dose has been declared.

[0077] As noted, a major potential adverse event commonly associated with TCE is cytokine release syndrome (CRS). The treatments with XPAT01, however, only resulted in CRS of low grades; hence no prophylactic steroid treatment was needed. Even if steroid is used, it is contemplated, relatively lower doses of a steroid would be sufficient. Such results, therefore, underscore the magnificent safety profiles of XPAT01.

[0078] In terms of efficacy, as shown in Example 3, not only was XPAT01 effective in treating HER2+cancers, it led to high percentages of responses even among those most challenging-to-treat tumors, e.g., ones that were resistant to 9 lines of prior therapies. Such prior therapies included HER2-targeted ones, such as trastuzumab (anti-HER2 antibody), trastuzumab deruxtecan (anti-HER2 ADC), tucatinib (small molecule HER2 inhibitor), and lapatinib (tyrosine kinase inhibitor).

[0079] Responses to the XPAT01 treatment can be evaluated with methods tested herein. For instance, tumor size reduction can be measured with CT scan. Biomarkers can also be used for this purpose, such as the increase of IFNyor decrease of circulating tumor DNA (ctDNA). Interestingly, certain biomarkers, such as decrease of levels of CEA (carcinoembryonic antigen), CA9-19, and CA-125, may precede tumor shrinkage and thus can serve as prognosis markers.

[0080] In some embodiments, the response may be measured with pharmacokinetic parameters, such as area under the concentration-time curve (AUC), maximum observed plasma concentration (Cmax). time to reach maximum concentration (tmax), accumulation ratio, and half life. In some embodiments, the response may be indicated with incidence of antidrug antibodies (AD As) at baseline with comparison to incidence of treatment-emergent AD As.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0081] Finally, pharmacokinetic / pharmacodynamics results show that XPAT01 has long half life which supports once weekly, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks or even once every eight weeks dosing.

[0082] 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 method entails administering to the patient a polypeptide of the instant disclosure, such as XPAT01 and variants, analogs and derivatives thereof, which arc described in further details below. Such polypeptides, containing an activatable TCE (T-cell engager) core having specificity to HER2 and CD3, are also referred to as XTENylated Protease-Activated bispecific T Cell Engager targeting HER2 (HER2-XPAT).

[0083] 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 HER2-XPAT.

[0084] In some embodiments, the HER2-XPAT is administered at a dose of at least 100 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 1 0 pg / kg of the HER2-XPAT. In some embodiments, each dose includes at least 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 HER2-XPAT. In some embodiments, each dose includes no more than 1500 pg / kg of the HER2-XPAT. In some embodiments, each dose includes no more than 1400 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 or 450 pg / kg of the HER2-XPAT.

[0085] In some embodiments, each dose includes from 150 pg / kg to 1500 pg / kg of the HER2-XPAT. In some embodiments, each dose includes from 200 pg / kg to 1300 pg / kg of the HER2-XPAT. In some embodiments, each dose includes from 300 pg / kg to 1200 pg / kg of the HER2-XPAT. In some embodiments, each dose includes from 400 pg / kg to 1000 pg / kg of the HER2-XPAT. In some embodiments, each dose includes from 450 pg / kg to 800 pg / kg of the HER2-XPAT.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO

[0086] In some embodiments, the HER2-XPAT is administered at a dose of 10.5 mg to 105 mg 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 10.5 mg of the HER2-XPAT. In some embodiments, each dose includes at least 11 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 or 100 mg of the HER2-XPAT. In some embodiments, each dose includes no more than 105 mg of the HER2-XPAT. In some embodiments, each dose includes no more than 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, or 11 mg of the HER2-XPAT.

[0087] In some embodiments, each dose includes from 10.5 mg to 105 mg of the HER2- XPAT. In some embodiments, each dose includes from 15 mg to 85 mg of the HER2-XPAT. In some embodiments, each dose includes from 20 mg to 55 mg of the HER2-XPAT. In some embodiments, each dose includes from 25 mg to 45 mg of the HER2-XPAT.In some embodiments, each dose includes from 28 mg to 42 mg of the HER2-XPAT. In some embodiments, each dose includes from 30 mg to 40 mg of the HER2-XPAT.

[0088] In some embodiments, the HER2-XPAT is administered once every week. In some embodiments, the HER2-XPAT is administered once every two weeks. In some embodiments, the HER2-XPAT is administered once every three weeks. In some embodiments, the HER2-XPAT is administered once every four weeks. In some embodiments, the IIER2-XPAT is administered once every five weeks. In some embodiments, the HER2-XPAT is administered once every six weeks. In some embodiments, the HER2-XPAT is administered once every seven weeks. In some embodiments, the HER2- XPAT is administered once every eight weeks. In some embodiments, the HER2-XPAT is administered once every nine weeks. In some embodiments, the HER2-XPAT is administered once every ten weeks.

[0089] Stepped-up dosing regimens have been tested in the accompanying experimental examples and exhibited excellent pharmacokinetic profiles (see, e.g., Example 2). The tested stepped-up dosing regimens included 100 / 200 / 400 pg / kg, 100 / 250 / 600 pg / kg, 100 / 300 / 800 pg / kg, 100 / 300 / 1000 pg / kg, 60 / 120 / 120 pg / kg, 60 / 120 / 200 pg / kg, 60 / 120 / 300 pg / kg, and 60 / 200 / 400 pg / kg,. Following each dose, only a minimal amount of the masked HER2-XPAT was unmasked outside the tumor environment (FIG. 2A), and the plasma concentrations of HER2-XPAT scaled linearly with the dose.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO

[0090] In accordance with one embodiment of the present disclosure, therefore, when the HER2-XPAT is administered multiple doses, a later dose can be higher than an earlier dose. For instance, in some embodiments, the HER2-XPAT is administered to a patient at (a) a first dose (also referred to as a first step-up dose), (b) a second dose (also referred to as a second step-up dose) later than and higher than the first dose, and (c) a third dose later than and higher than the second dose. It is important to note that the first and second doses are not necessarily adjacent doses; neither are second and third doses. In some embodiments, the third dose is the target dose, when no additional dose escalation is intended (i.e., this is the dose amount to be administered to the patient in the second dosing cycle and any subsequent dosing cycles). In some embodiments, third dose is a third step-up dose if an additional dose escalation is required to reach the target dose.

[0091] In some embodiments, the second dose is at least 10% higher than the first dose. In some embodiments, the second dose is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%. 160%, 170%, 180%. 190%, 200%, 210%. 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390% or 400% higher than the first dose.

[0092] In some embodiments, the third dose is at least 10% higher than the second dose. In some embodiments, the third dose is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390% or 400% higher than the second dose.

[0093] In some embodiments, the third dose is at least 10% higher than the first dose. In some embodiments, the third dose is at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times 10 times, 12 times, 15 times or 20 times of the first dose.

[0094] In some embodiments, the second dose is at least a week after the first dose. In some embodiments, the second dose is at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks or 12 weeks after the first dose. In some embodiments, the third dose is at least a week after the second dose. In some embodiments, the third dose is at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after the second dose. In some embodiments, theVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO third dose is at least 2 weeks after the first dose. In some embodiments, the third dose is at least 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks 17 weeks, 18 weeks, 19 weeks or 20 weeks after the first dose.

[0095] In some embodiments, there is no additional dose between the first dose and the second dose (i.e., they are consecutive doses of the HER2-XPAT). In some embodiments, there is no additional dose between the second dose and the third dose (i.e., they are consecutive doses of the HER2-XPAT). In some embodiments, there is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 additional doses between the first dose and the second dose. In some embodiments, there is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 additional doses between the second dose and the third dose.

[0096] In some embodiments, the third dose includes at least 400 |Jg / kg of the HER2-XPAT. In some embodiments, the third dose includes at least 450 |Jg / kg, 500 |Jg / 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 HER2- XPAT. In some embodiments, the third dose includes no more than 1500 pg / kg of the HER2- XPAT. In some embodiments, each dose includes no more than 1400 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 or 450 pg / kg of the HER2-XPAT.

[0097] In some embodiments, the third dose includes from 400 pg / kg to 1500 pg / kg of the HER2-XPAT. In some embodiments, the third dose includes from 500 pg / kg to 1400 pg / kg of the HER2-XPAT. In some embodiments, the third dose includes from 600 pg / kg to 1200 pg / kg of the HER2-XPAT. In some embodiments, the third dose includes from 700 pg / kg to 1100 pg / kg of the HER2-XPAT. In some embodiments, the third dose includes from 800 pg / kg to 1000 pg / kg of the HER2-XPAT.

[0098] In some embodiments, the third dose includes at least 28 mg of the HER2-XPAT. In some embodiments, the third dose includes at least 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 or 100 mg of the HER2- XPAT. In some embodiments, the third dose includes no more than 105 mg of the HER2- XPAT. In some embodiments, the third dose includes no more than 100 mg, 95 mg, 90 mg,Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO85 mg, 80 mg, 75 mg, 70 mg, 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, or 35 mg of the HER2-XPAT.

[0099] In some embodiments, the third dose includes from 28 mg to 105 mg of the HER2- XPAT. In some embodiments, the third dose includes from 35 mg to 98 mg of the IIER2- XPAT. In some embodiments, the third dose includes from 42 mg to 84 mg of the HER2- XPAT. In some embodiments, the third dose includes from 49 mg to 77 mg of the HER2- XPAT.In some embodiments, the third dose includes from 56 mg to 70 mg of the HER2- XPAT.

[0100] In some embodiments, the HER2-XPAT is administered to a patient in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle. In some embodiments, the first dosing cycle comprises stepped-up dosing, wherein two or three doses of the HER2-XPAT are administered prior to a target dose of the HER2-XPAT in increasing amounts, but less than the target dose. In some embodiments, the second dosing cycle does not comprise stepped-up dosing. Tn some embodiments, the second dosing cycle comprises administration to the patient of the target dose. In some embodiments, the dosing regimen further comprises one, two, three, four, five, six, seven, eight, nine, or ten additional dosing cycles. In some embodiments, the additional dosing cycles are identical to the second dosing cycle. In some embodiments, the second dosing cycle, and any subsequent dosing cycles comprises or consists of a single administration to the patient of the target dose.

[0101] In some embodiments, the first step-up dose, the second step-up dose, and optionally, the third step-up dose are administered to the patient approximately 6-8 days apart. In some embodiments, the target dose in the first dosing cycle is administered 6-8 days after the final step-up dose. In some embodiments, the first dosing cycle can be 21, 28, 35, or 42 days in length.

[0102] In some embodiments, a target dose of the HER2-XPAT is administered to the patient on the first day of the second dosing cycle. In some embodiments, the second dosing cycle can be 21, 28, 35, 42, 49, 52, or 63 days in length.

[0103] Combination therapies have also been tested for HER2-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 instantVir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO disclosure (e.g., HER2-XPAT) and 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 a polypeptide of the instant disclosure (e.g., HER2-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., HER2- XPAT).

[0104] 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.

[0105] Pembrolizumab (formerly MK-3475 or lambrolizumab, Keytruda) is an anti-PD-1 monoclonal antibody developed by Merck and first approved by the Food and Drug 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.

[0106] 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.

[0107] 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.

[0108] Spartalizumab (PDR001) is an anti-PD-1 monoclonal antibody developed by Novartis to treat both solid tumors and lymphomas.

[0109] Camrelizumab (SIIR1210) is an anti-PD-1 monoclonal antibody introduced by Jiangsu HengRui Medicine Co., Ltd. that recently received conditional approval in China for the treatment of relapsed or refractory classical Hodgkin lymphoma.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0110] 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).

[0111] Tislelizumab (BGB-A317) is a humanized IgG4 anti-PD-1 monoclonal antibody developed by BeiGene for solid tumors and hematologic cancers.

[0112] Dostarlimab (TSR-042, WBP-285) is a humanized monoclonal antibody against PD-1 under investigation by GlaxoSmithKline.

[0113] INCMGA00012 (MGA012) is a humanized IgG4 monoclonal antibody developed by Incyte and MacroGenics.

[0114] AMP-224 is an anti-PD-1 monoclonal antibody by AstraZeneca / Medlmmune and GlaxoSmithKline.

[0115] AMP-514 (MEDI0680) is an anti-PD-1 monoclonal antibody by AstraZeneca.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] KN035 is an anti-PD-Ll antibody with subcutaneous formulation currently under clinical evaluations in the US, China, and lapan.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO

[0121] CK-301 is an anti-PD-Ll antibody being developed by Checkpoint Therapeutics.

[0122] Some small peptide and small molecule inhibitors are also being developed. Examples are shown below.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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. In some embodiments, the cancer is characterized with PD-L1 expression in the tumor.

[0127] In a combination therapy, in some embodiments, the HER2-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 HER2-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 HER2-XPAT. In some embodiments, each dose includes no more than 1000 pg / kg of the HER2-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 HER2-XPAT.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0128] In some embodiments, each dose includes from 30 pg / kg to 1000 pg / kg of the HER2- XPAT. In some embodiments, each dose includes from 40 pg / kg to 500 pg / kg of the HER2- XPAT. In some embodiments, each dose includes from 45 pg / kg to 200 pg / kg of the HER2- XPAT. In some embodiments, each dose includes from 45 pg / kg to 120 pg / kg of the HER2- XPAT. In some embodiments, each dose includes from 50 pg / kg to 100 pg / kg of the HER2- XPAT. In some embodiments, each dose includes from 50 pg / kg to 70 pg / kg of the HER2- XPAT. In some embodiments, each dose includes from 55 pg / kg to 65 pg / kg of the HER2- XPAT.

[0129] In a combination therapy, in some embodiments, the HER2-XPAT is administered at a dose of 2.1 mg to 70 mg 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 2.1 mg of the HER2-XPAT. In some embodiments, each dose includes at least 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 11 mg, 12 mg, 13 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg or 60 mg of the HER2- XPAT. In some embodiments, each dose includes no more than 70 mg of the HER2-XPAT. In some embodiments, each dose includes no more than 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, 14 mg, 13 mg, 12 mg, 11 mg, 10 mg, 9 mg, 8.5 mg, 8 mg, 7.5 mg, 7 mg, 6.5 mg, 6 mg, 5.5 mg, 5 mg, 4.5 mg, 4 mg, 3.5 mg or 3 mg of the HER2-XPAT.

[0130] In some embodiments, each dose includes from 2.1 mg to 70 mg of the IIER2-XPAT. In some embodiments, each dose includes from 2.8 mg to 35 mg of the HER2-XPAT. In some embodiments, each dose includes from 3.2 mg to 14 mg of the HER2-XPAT. In some embodiments, each dose includes from 3.2 mg to 8.4 mg of the HER2-XPAT. In some embodiments, each dose includes from 3.5 mg to 7 mg of the HER2-XPAT. In some embodiments, each dose includes from 3.5 mg to 4.9 mg of the HER2-XPAT. In some embodiments, each dose includes from 3.8 mg to 4.5 mg of the HER2-XPAT.

[0131] In some embodiments, the HER2-XPAT is administered once every week. In some embodiments, the HER2-XPAT is administered once every two weeks. In some embodiments, the HER2-XPAT is administered once every three weeks. In some embodiments, the HER2-XPAT is administered once every four weeks. In some embodiments, the HER2-XPAT is administered once every five weeks. In some embodiments, the HER2-XPAT is administered once every six weeks. In some embodiments,Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO the HER2-XPAT is administered once every seven weeks. In some embodiments, the HER2- XPAT is administered once every eight weeks. In some embodiments, the HER2-XPAT is administered once every nine weeks. In some embodiments, the HER2-XPAT is administered once every ten weeks.

[0132] In accordance with one embodiment of the present disclosure, therefore, when the HER2-XPAT is administered multiple doses in a combination therapy, a later dose can be higher than an earlier dose. For instance, in some embodiments, the HER2-XPAT is administered to a patient at (a) a first dose, (b) a second dose later than and higher than the first dose, and (c) a third dose later than and higher than the second dose. It is important to note that the first and second doses are not necessarily adjacent doses; neither are second and third doses.

[0133] In some embodiments, the second dose is at least 10% higher than the first dose. In some embodiments, the second dose is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390% or 400% higher than the first dose.

[0134] In some embodiments, the third dose is at least 10% higher than the second dose. In some embodiments, the third dose is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390% or 400% higher than the second dose.

[0135] In some embodiments, the third dose is at least 10% higher than the first dose. In some embodiments, the third dose is at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times 10 times, 12 times, 15 times or 20 times of the first dose.

[0136] In some embodiments, the second dose is at least a week after the first dose. In some embodiments, the second dose is at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after the first dose. In some embodiments, the third dose is at least a week after the second dose. In some embodiments, the third dose is at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after the second dose. In some embodiments, theVir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO third dose is at least 2 weeks after the first dose. In some embodiments, the third dose is at least 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks 17 weeks, 18 weeks, 19 weeks or 20 weeks after the first dose.

[0137] In some embodiments, there is no additional dose between the first dose and the second dose (i.e., they are consecutive doses of the HER2-XPAT). In some embodiments, there is no additional dose between the second dose and the third dose (i.e., they are consecutive doses of the HER2-XPAT). In some embodiments, there is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 additional doses between the first dose and the second dose. In some embodiments, there is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 additional doses between the second dose and the third dose.

[0138] In some embodiments, the third dose includes from 50 |ig / kg to 1000 |ig / kg of the HER2-XPAT. In some embodiments, the third dose includes from 60 pg / kg to 500 pg / kg of the HER2-XPAT. In some embodiments, the third dose includes from 70 pg / kg to 400 pg / kg of the HER2-XPAT. In some embodiments, the third dose includes from 80 pg / kg to 300 pg / kg of the HER2-XPAT. In some embodiments, the third dose includes from 90 pg / kg to 200 pg / kg of the HER2-XPAT. In some embodiments, the third dose includes from 100 pg / kg to 180 pg / kg of the HER2-XPAT. In some embodiments, the third dose includes from 120 pg / kg to 150 pg / kg of the HER2-XPAT.

[0139] In a combination therapy, in some embodiments, the third dose includes at least 3.5 mg of the HER2-XPAT. In some embodiments, the third dose includes at least 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 11 mg, 12 mg, 13 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg or 60 mg of the HER2-XPAT. In some embodiments, the third dose includes no more than 70 mg of the HER2-XPAT. In some embodiments, the third dose includes no more than 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, 14 mg, 13 mg, 12 mg, 11 mg, 10 mg, 9 mg, 8.5 mg, 8 mg, 7.5 mg, 7 mg, 6.5 mg, 6 mg, 5.5 mg, 5 mg, or 4.5 mg of the HER2-XPAT.

[0140] In some embodiments, the third dose includes from 3.5mg to 70 mg of the HER2-XPAT. In some embodiments, the third dose includes from 4.2 mg to 35 mg of the HER2-XPAT. In some embodiments, the third dose includes from 4.9 mg to 28 mg of the HER2-Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOXPAT. In some embodiments, the third dose includes from 5.6 mg to 21 mg of the HER2- XPAT. In some embodiments, the third dose includes from 6.3 mg to 14 mg of the HER2- XPAT. In some embodiments, the third dose includes from 7 mg to 12.6 mg of the HER2- XPAT. In some embodiments, the third dose includes from 8.4 mg to 10.5 mg of the HER2- XPAT.

[0141] Combination dosing regimens are also provided, in some embodiments. One example combination dosing regimen includes the administration of HER2-XPAT and an anti-PDLl or anti-PDl inhibitor, such as pcmbrolizumab.

[0142] In some embodiments, HER2-XPAT is administered according to a dosing regimen as described above. For instance, in some embodiments, the HER2-XPAT dosing regimen includes at least a first dosing cycle and a second dosing cycle. In some embodiments, the first dosing cycle comprises stepped-up dosing, wherein two or three doses of the HER2- XPAT are administered prior to a target dose of the HER2-XPAT in increasing amounts, but less than the target dose. In some embodiments, the second dosing cycle does not comprise stepped-up dosing. In some embodiments, the second dosing cycle comprises administration to the patient of the target dose. In some embodiments, the dosing regimen further comprises one, two, three, four, five, six, seven, eight, nine, or ten additional dosing cycles. In some embodiments, the additional dosing cycles are identical to the second dosing cycle. In some embodiments, the second dosing cycle, and any subsequent dosing cycles comprises or consists of a single administration to the patient of the target dose.

[0143] In some embodiments, the first step-up dose, the second step-up dose, and optionally, the third step-up dose are administered to the patient approximately 6-8 days apart. In some embodiments, the target dose in the first dosing cycle is administered 6-8 days after the final step-up dose. In some embodiments, the first dosing cycle can be 21, 28, 35, or 42 days in length.

[0144] In some embodiments, during the first dosing cycle and the second dosing cycle of HER2-XPAT, the anti-PDLl or anti-PDl inhibitor, such as pembrolizumab, is also administered. In some embodiments, the pembrolizumab is administered at 200 mg once every three weeks, or 400 mg once every six weeks. In some embodiments, pembrolizumab is administered in any cycle when HER2-XPAT is administered.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOCancer Treatments and Treatment Monitoring

[0145] The compositions, methods and treatment regimens are useful for treating various cancer types, in particular those characterized with HER2 expression.

[0146] HER2 (receptor tyrosine-protein kinase erbB-2) is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. HER2 activation results from heterodimerization with another ERBB member or by homodimerization when HER2 concentration are high, for instance in cancer. Amplification, also known as the overexpression of the HER2 gene, occurs in approximately 15-30% of breast cancers. HER2- positive breast cancers are well established as being associated with increased disease recurrence and a poor prognosis.

[0147] HER2 expression or over-expression is also known to occur in ovarian, stomach, adenocarcinoma of the lung and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma, e.g., HER2 is over-expressed in approximately 7-34% of patients with gastric cancer and in 30% of salivary duct carcinomas.

[0148] HER2 expression 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.

[0149] Immunohistochemistry (IHC) is generally used to measure the amount of HER2 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. HER2 IHC ScoresVir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0150] Fluorescence in situ hybridization (FISH) can also be used to measure the number of copies of the gene which are present. It usually uses chromosome enumeration probe 17 (CEP17) to count the amount of chromosomes. Hence, the HER2 / CEP17 ratio reflects any amplification of HER2 as compared to the number of chromosomes. The signals of 20 cells arc usually counted. A sample classification system is provided in Table B.Table B. FISH Classification of HER2

[0151] It is becoming increasingly clear that the aberrations seen in HER2 signaling and expression are a driver of oncogenesis and a resistance pathway for various treatments in many tumors. The type of HER2 aberration may be tumor dependent. For example, in colorectal cancer (CRC), patients treated with prior epidermal growth factor receptor (EGFR) inhibitors may develop HER2 amplification, whereas non-small-cell lung cancer (NSCLC) patients predominantly develop activating mutations in HER2, especially after prior therapies such as EGFR inhibitors.

[0152] Breast cancer is one the most common cancers globally, with 1.7 million cases diagnosed. While major advances have been made with effective HER2-directed treatments, patients who experience disease recurrence or develop metastatic disease are incurable and will eventually succumb to their disease. In the metastatic front-line setting, the standard of care for HER2+ breast cancer is trastuzumab and pertuzumab in combination with taxanes; in later-line treatment settings, there are multiple chemotherapies that are used in combination with HER2-targeted biologies (trastuzumab, margetuximab) or HER2 TKTs (tucatinib, lapatinib, neratinib); additionally, there are options for ADCs such as TDM-1, or famtrastuzumab deruxtecan (National Comprehensive Cancer Network [NCCN] 2021). To date, no checkpoint inhibitors (CPIs) or cancer immunotherapies have shown added survival benefit in this disease. Newer therapies, especially immunotherapies with the potential for long lasting immunity and OS benefit, are needed.

[0153] While gastric cancer and GEI cancer mortality rates have declined in recent decades, more than one million people are diagnosed each year and gastric / GEI cancers remain theVir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO third-leading cause of cancer-related death. The major improvements in mortality rates have been primarily due to improved screening and aggressive surgical resections in countries such as Japan. In the United States and Europe, gastric cancer is often diagnosed in the advanced and unresectable stage with short survival times. In gastric and GEJ tumors that are unrcscctablc or metastasized, the prognosis remains poor, with survival often 1 year or less with chemotherapy. HER2 is a validated target and is overexpressed in about 20% to 30% of all gastric / GEJ tumors. Currently, trastuzumab is the only drug approved in the first-line setting, in combination with fluoropyrimidine and cisplatin-based therapy. Other HER2- targeted agents, such as pertuzumab, lapatinib, and T-DM1, that have been approved in HER2+ breast cancer have not demonstrated significant efficacy in gastric / GEJ cancer. One reason for this discrepancy may be that HER2 staining in gastric cancer is heterogeneous.

[0154] While the role of HER2 in breast and gastric cancer has been well established for many years, only recently has there been a recognition of the importance of HER2 in other disease types, in particular as a resistance biomarker to other signaling pathways such as EGFR. With more patients undergoing biopsies after progression from prior therapies, and the advance of more routine molecular genotyping, numerous aberrations in HER2 have been discovered. These include IIER2 overexpression, amplification, and activating mutations in a large variety of tumors, including, but not limited to, CRC, NSCLC, biliary, ovarian, endometrial, salivary, sarcomas and other cancers. In CRC, HER2 amplification and overexpression has been found to be a resistance mechanism to cetuximab.

[0155] In NSCLC, activating HER2 mutations have been identified with or without concomitant HER2 overexpression and amplification. The most common mutation is an exon 20 in-frame mutation that has been shown to be activating and oncogenic. It occurs in approximately 2% to 4% of NSCLC, and more predominantly in Asian nonsmokers, similar to EGFR mutations. It is also found to occur in both EGFR-mutant and EGFR- wild-type tumors. Fam-trastuzumab deruxtecan has recently shown promising activity in HER2-mutant NSCLC, even with HER2 having low expression (immunohistochemistry [IHC] 1 to 2+).

[0156] In some embodiments, the patient has a HER2+cancer. In some embodiments, the HER2+cancer has an immunohistochemistry (IHC) grade 2+. In some embodiments, the HER2+cancer has an immunohistochemistry (IHC) grade 3+. In some embodiments, the HER2+cancer is in situ hybridization positive (ISH+). In some embodiments, the HER2+cancer has one or more HER2 mutations.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO

[0157] 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.

[0158] 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.

[0159] 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- HER2 treatment. In some embodiments, the anti-HER2 therapy comprises one or more of an anti-HER2 antibody or small molecule inhibitor, such as trastuzumab, trastuzumab deruxtecan, tucatinib or lapatinib.

[0160] In some embodiments, the cancer patient is a breast cancer patient that has undergone or is resistant to at least one line of prior treatments. In some embodiments, the cancer patient has gastric cancer or gastroesophageal junction cancer and has undergone or is resistant to at least one line of prior treatments. In some embodiments, the cancer patient has metastatic non-small cell lung cancer (NSCLC) or metastatic colorectal cancer (CRC) and hasVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO progressed on a prior treatment. In some embodiments, the CRC is characterized as microsatellite stable (MSS).

[0161] 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 HER2-XPAT, the patient is not administered a prophylactic dose of a steroid. In some embodiments, during the treatment with the HER2-XPAT, the patient is not also treated with a steroid.

[0162] In some embodiments, even when a steroid 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. In some embodiments, the dexamethasone is used only once.

[0163] 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 IL-6 expression is detected.

[0164] 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 CEA (carcinoembryonic antigen) level is detected, e.g., in the blood or another body fluid, it can be predicted that the patient is responding to the HER2-XPAT treatment and tumor shrinkage will follow. In some embodiments, the biomarker is ctDNA (circulating tumor DNA), CA - 19, or CA-125.HER2-XPAT

[0165] An example polypeptide suitable for use in the instant methods is HER2-XPAT, which contains a TCE (T-cell engager) core having dual specificity to HER2 and CD3, flanked by a N-terminal and a C-terminal unstructured polypeptide masks (XTENs).

[0166] “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 atVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO 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 whole antibodies or antibody fragments (e.g. single-chain antibodies and Fc fragments). Examples of XTEN can be found in W02017040344, without limitation.

[0167] In one embodiment, the HER2-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-HER2 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 protease-cleavable site, and (b) is connected to (c) through a second protease-cleavable site.

[0168] 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.

[0169] In some embodiments, the anti-HER2 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.

[0170] In some embodiments, the anti-HER2 VH and the anti-HER2 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.

[0171] In some embodiments, the anti-HER2 antigen-binding fragment has a VL-linker-VH configuration. In some embodiments, the anti-HER2 antigen-binding fragment has a VH- linker-VL configuration. In some embodiments, the anti-HER2 antigen-binding fragment hasVir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:13.

[0172] In some embodiments, the anti-CD3 antigen-binding fragment include a heavy chain variable region (VII) and a light chain variable region (VL). In some embodiments, the VII 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] In some embodiments, the anti-HER2 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- HER2 antibody or antigen-binding fragment, the linker, and the anti-CD3 antibody or antigen-binding fragment, from N-terminus to C-terminus. In some embodiments, the T-cell engager core includes the anti-CD3 antibody or antigen-binding fragment, the linker, and the anti-HER2 antibody or antigen-binding fragment, from N-terminus to C-terminus.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0177] 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.

[0178] 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 NOT E

[0179] In some embodiments, the HER2-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 HER2-XPAT is XPAT01 which includes the sequence of SEQ ID NO: 1.Pharmaceutical Compositions

[0180] Disclosed herein includes a pharmaceutical composition comprising a polypeptide, such as the HER2-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.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0181] 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, 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.

[0182] 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.

[0183] In certain embodiments, the polypeptide of the instant disclosure (e.g., HER2-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 preferred second or additional therapeutic agents include other HER2 targeting agents, chemotherapy agents, radiotherapeutic agents, as well as agents that target HER3 and other targets that are involved in resistance to treatment of HER2-driven cancers.

[0184] In other embodiments, the additional agent may be a DNA damaging agent, antimetabolite, anti-microtubule agent, antibiotic agent, etc. DNA damaging agents include 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 asVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO 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 ixabcpilonc (Ixcmpra®). Antibiotic agents include without limitation actinomycin, anthracyclines, valrubicin, epirubicin, bleomycin, phcamycin, mitomycin, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.

[0185] 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, anthracyclines, valrubicin, epirubicin, bleomycin, plicamycin, mitomycin, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.

[0186] 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 include 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 series, Genentech (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), IPI-145 (Intellikine Inc.), KAR-4139Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO(Karns Therapeutics, Chilworth, UK), KAR-4141 (Karns Therapeutics), KIN-1 (Karus Therapeutics), KT 5720 (CAS #108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS #1032350-13-2), ML-9 (CAS #105637-50-1), Naltrindole Hydrochloride, OXY-111A (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, Genentech (Roche Holdings Inc.), PI3 kinase delta inhibitors, Incozen (Incozen 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 Evotcc (Evotcc), 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.), 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.

[0187] 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.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0188] 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.

[0189] 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.

[0190] 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. Antiangiogenesis 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

[0191] Multiple studies were conducted to characterize the pharmacology, pharmacokinetics (PK), pharmacodynamics, and toxicity of the investigational prodrug and clinical candidate, XPAT01.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0192] XPAT01 (SEQ ID NO:1, see Table 1) is a human epidermal growth factor receptor 2 (HER2) -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 HER2. 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 cither HER2 or CD3. Protease cleavage sites (SEQ ID NO:3) 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 SequencesVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WONonclinical Pharmacology

[0193] Multiple in vitro studies have been conducted to characterize the binding and biological activities of XPAT01 and its metabolites. When proteolytically activated, the activated TCE (XPAT01-TCE) bound with low nanomolar affinities to human HER2 and CD3 and was cross-reactive with cynomolgus monkeys, supporting the use of cynomolgus monkey as a species for toxicity and PK studies.

[0194] In the requisite presence of both HER2 and T cells, XPAT01-TCE mediated highly potent, target-directed cytotoxicity, T-cell activation, and cytokine secretion at low picomolar concentrations. In contrast, in its masked form and in the absence of protease dysregulation, XPAT01 provided 3 to 4 orders of magnitude fold protection against target cell killing and T- cell activation (Table 2).Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOTable 2. Equilibrium Binding (Kd) for XPAT01 and Its Proteolytic Metabolites to Human and Cynomolgus Monkey HER2 and CI)3i

[0195] In vitro, the majority of inflammatory cytokines were not significantly co-induced from human or cynomolgus monkey peripheral blood mononuclear cells (PBMCs) by XPAT01 and XPAT01-TCE at concentrations relevant for in vivo activity.

[0196] In vivo, XPAT01 induced complete tumor regressions in the HER2 high BT-474 breast tumor model (-975,000 HER2 receptors) that equaled those observed with equimolar dosing of the unmasked XPAT01-TCE. XPAT01 was also highly efficacious in the HER2- low HT-55 colorectal model that expressed only 25,000 surface HER2 receptors. Efficacy of XPAT01 was dependent on cleavage at its protease release site and was associated with activation of intratumoral T cells, while in circulation, XPAT01 remained proteolytically stable and T cells remained quiescent.

[0197] Preferential unmasking of a fluorophore-labeled XPAT01 was evident in tumors following a 2-day incubation in BT-474 tumor-bearing mice compared to heart, brain and liver tissue combined, supporting the key tenets of localized dysregulation of proteases in tumors and the dominance of protease inhibition in normal tissues.In vivo Efficacy StudiesI. Complete tumor regressions in mice bearing HER2 BT-474 tumors and engrafted with human peripheral blood mononuclear cells (hPBMCs) were seen with XPAT01 in a cleavage-dependent manner.

[0198] In mice bearing established HER2 BT-474 tumors and engrafted with hPBMCs, administration of three doses of XPATOl or its prototype XPAT0 IP 1 at 2.1 mg / kg (QW, 3x) induced complete tumor regression (129% tumor growth inhibition [TGI] for XPAT01 andVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO111% TGI for XPAT01P1; both p < 0.001), similar to the efficacy obtained with equimolar doses of unmasked XPAT01-TCE (133% TGI; p < 0.001). In this study, NOD / ShiscidIL2rgammanu11(NOG) mice were inoculated SC with 2xl07BT-474 tumor cells, engrafted with IxlO7hPBMCs and treated 2 days later with the indicated test articles at equimolar doses QW for 3 weeks.

[0199] XPAT01, 818-P1 and XPAT01-TCE induced comparable and highly significant tumor growth inhibition, including complete tumor regression in most mice (with unpalpable tumors). Lack of tumor growth inhibition by the XPATOl-NoClvSitc demonstrates the requirement of protease cleavage for efficacy. The robust efficacy was protease dependent, as demonstrated by the lack of significant TGI in mice treated with XPAT01 without proteolytic cleavage site (NoClvSite), which showed growth curves similar to mice in the vehicle control group (FIG. 1).II. Complete tumor regressions in mice bearing HER2-low HT-55 tumors and engrafted with hPBMCs are seen with higher XPAT01 doses

[0200] In mice engrafted with hPBMC and bearing low HER2 colorectal HT-55 tumors, administration of XPAT01 at 5.1 mg / kg induced complete tumor regression in all mice (103% TGI; p < 0.01), similar to the response observed with XPAT01-TCE (105% TGI; p < 0.01). The robust anti-tumor activity was dose dependent, since XPAT01 at 2.1 mg / kg induced 70% TGI (p value nonsignificant), as well as protease dependent, again demonstrated by the lack of efficacy when mice are treated with the same dose of XPATOl-NoClvSite (- 27% TGI).III. Combination efficacy of XPAT01 and pembrolizumab in the treatment of BT-474 tumor in NSG mice engrafted with hPBMCs

[0201] In the BT-474 model, administration of QW 0.5 mg / kg XPAT01 (a total of three doses) and twice-weekly 10 mg / kg pembrolizumab (a total of six doses) exhibited enhanced TGI compared to that of either single agent alone (FIG. 8). The combination TGI of 99% was statistically significant (p < 0.0001) compared to XPAT01 or pembrolizumab alone, with TGI of 74% and 30%, respectively (p < 0.0001 and p < 0.008, respectively). The combination was well tolerated as demonstrated by the lack of body weight loss in all groups of mice that received either agent as single agent or in combination. These data support clinical evaluation of XPAT01 in combination with pembrolizumab.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOIn Vivo Toxicology StudiesI. In cynomolgus monkeys, the maximum tolerated dose (MTD) for the clinical candidate, XPAT01 was 42 mg / kg, and the MTD for unmasked XPAT01-TCE was 0.2 mg / kg, translating to a safety margin of greater than 400-fold (based on maximum plasma concentration (Cmax) between masked and unmasked molecules

[0202] XPAT01 was well tolerated up to a single dose of 42 mg / kg; in contrast, the MTD for XPAT01-TCE was 0.2 mg / kg / day following continuous intravenous (IV) infusion. The toxicities of XPAT01-TCE were consistent with cytokine release with significant elevation of cytokines. As anticipated, the XTEN masks allowed for a higher dose (88-fold higher molar dose / kg [298 nm / kg for XPAT at 42 mg / kg and 3.37 nm / kg for PAT] at 0.2 mg / kg / day) of the XPAT01 prodrug to be administered compared with the fully activated XPAT01 -TCE.II. Mild elevations of cytokines at the XPAT01 MTD of 42 mg / kg did not correlate with clinical manifestations of cytokine release syndrome (CRS)

[0203] Dose-dependent XPAT01 -related findings included decreased lymphocytes, increased neutrophils, increased C-reactive protein (CRP), and CD4+ T cell activation. There were minimal and subclinical elevations of cytokines at doses up to 42 mg / kg (excluding an animal with an infusion reaction). At 50 mg / kg of XPAT01-P1, IL-6, monocyte chemoattractant protein l(MCP-l), and tumor necrosis factor alpha (TNF-a) were mildly elevated, but this did not correlate with obvious clinical manifestations of cytokine release.

[0204] In all studies with cynomolgus monkeys, elevations in creatine kinase (CK) were frequently observed within the first 24 hours of infusion, but with no relationship to dose; this is consistent with known skeletal muscle injury associated with animal handling and restraint during infusion of test articles. CK isoforms were also evaluated and suggested a noncardiac source. Aspartate aminotransferase (AST) without alanine aminotransferase (ALT) elevations also correlated with CK elevations, which suggested a non-liver source of AST. In addition, there were no histopathological correlates in skeletal, cardiac, or liver tissue in either single- or multiple-dose studies when necropsy was performed.Pharmacokinetic and Toxicokinetic Studies

[0205] The PK and toxicokinetics (TK) of XPAT01 and its metabolites after single- and repeat-dose administration was evaluated in multiple studies in cynomolgus monkeys at doses ranging from 0.5 mg / kg to 42 mg / kg. XPAT01 demonstrated a biphasic PK profileVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO following single- and repeat-dose IV administration, with dose proportional PK observed across all evaluated dose levels. Based on PK modeling using observed data from cynomolgus monkeys administered XPAT01 (0.5 mg / kg to 42 mg / kg), the estimated typical total clearance (CL), volume of distribution at steady-state (Vss) and tl / 2 for a 3 kg cynomolgus monkey were approximately 26.2 mL / day, 155 mL, and 2.92 days, respectively. A 67% incidence of AD As (24 of 36 animals administered 0.5 mg / kg to 6 mg / kg XPAT01) was observed during the treatment phase of the repeat dose GLP cynomolgus monkey study, with a reduced systemic exposure of XPAT01 in a subset of ADA-positive animals compared to ADA-negative animals.

[0206] The metabolism and peripheral stability of XPAT01 was assessed in vivo in tumorbearing mice and cynomolgus monkeys and in vitro in plasma samples from cynomolgus monkeys (healthy monkeys and monkeys with drug-induced systemic inflammation) and humans (healthy volunteers, cancer patients, patients with inflammatory diseases). Data from in vivo studies support that XPAT01 remains largely intact in systemic circulation, with low to negligible levels of metabolites detected in systemic circulation. In tumor-bearing mice, consistent with the design of XPAT01 to be preferentially cleaved in the tumor, metabolites of XPAT01 (XPATOl(lx-N), XPATOl(lx-C) and XPAT01-TCE), were detected at a higher metabolite to parent ratio in tumor than in peripheral tissues or plasma. In vitro plasma stability experiments demonstrate that XPAT01 has a similar relative abundance profile of cleavage products in human and cynomolgus monkey plasma and suggest that the cleavage of XPAT01 in plasma does not appear to be dramatically enhanced in diseased conditions. Collectively, these data along with binding data demonstrating that XPAT01 and its fully unmasked metabolite.Example 2. Clinical Evaluation of XPAT01

[0207] This is a Phase 1, first-in-human (FIH) study for XPAT01 in patients with HER2- expressing solid tumors.

[0208] Part 1 includes a dose escalation of single-agent XPAT01 on a QW schedule and a dose escalation on an every-3-week (Q3W) schedule supported by the initial PK analyses and clinical experience. Part 2 is a Phase lb dose escalation of XPAT01 in combination with pembrolizumab. Parts 3 and 4 are Phase lb expansion cohorts in multiple indications,Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO evaluating single-agent XPAT01 (Part 3) or in combination with pembrolizumab (Part 4), respectively.

[0209] Tumor types evaluated include those overexpressing HER2, harboring activating mutations of IIER2, such as IIER2+or in situ hybridization-positive (ISII+) breast cancer, HER2-expressing gastroesophageal junction / gastric cancer, hormone receptor-positive (HR+) breast cancer, colorectal cancer, non-small-cell lung cancer (NSCLC), salivary gland cancer, and a tumor agnostic basket of rare tumor types with HER2 aberrations (Table 3).Clinical Study ProtocolInclusion Criteria

[0210] Participants must meet certain criteria to be included in the study, such as written informed consent. Diseases under study, prior lines of therapy, and HER2 status, per local tests, are shown in Table 3.Table 3. Inclusion CriteriaVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOAbbreviations: CAP, College of American Pathologists; CDK, cyclin-dependent kinase; CDx, companion diagnostic; CLIA, Clinical Laboratory Improvement Amendments; CRC, colorectal cancer; DISH, dual in situ hybridization; FISH, fluorescence in situ hybridization; GEJ, gastroesophageal junction; HR+, hormone receptor positive; IHC, immunohistochemistry; ISH, in situ hybridization; ISO, International Organization for Standardization; mCRC, metastatic colorectal cancer; mNSCLC, metastatic non-small-cell lung cancer; NGS, next-generation sequencing; NSCLC, non-small-cell lung cancer.aKnown HER2 status strongly preferred; in Parts 3 and 4, prior approved HER2-directed therapy may be used instead of HER2 status report, with approval from Sponsor medical monitor.bIHC: as determined by a CLIA / CAP or ISO17025 / ISO15189-compliant or equivalent-accredited HER2 testcISH positivity defined as ratio of > 2.0 for the number of HER2 gene copies to the number of signals for CEP17.dISH includes HSH / DISH.eHER2-activating mutation per NGS testing (tumor tissue), including, but not limited to, exon 20 insertionsfNGS (tumor tissue based): as determined by a CLIA / CAP or ISO17025 / ISO15189-compliant or equivalent- accredited NGS AssaygFor determination of HER2 low status by IHC: The Ventana PATHWAY Assay should be used and scoring performed in accordance with the local diagnostic package insert’s interpretation manualFormulation, Packaging, and Labeling

[0211] XPAT01 drug product for injection was supplied as a single -use, sterile, white to off- white lyophilized powder for solution for IV infusion in glass vials. After reconstitution with an appropriate volume of Sterile Water for Injection, EP / USP as indicated in the Pharmacy Manual, each mL of XPAT01 solution contains 4 mg of XPAT01 .Clinical Pharmacokinetics

[0212] Preliminary PK data have been obtained from 75 participants treated with 1 to 1000 yg / kg QW XPAT01 or Q3W with QW step-up (Part 1). Plasma concentration-time profiles of XPAT01 following the first (Cycle 1, Day 1 [C 1D1 ]) and third (C1D15) were calculated for fixed and step-up regimens. Preliminary PK parameters following the first (C1D1) and third (CID 15) dose were calculated using noncompartmental analyses and are provided in Tables 4 and 5 for fixed and step-up regimens (FIG. 9A-B), respectively. Cmax and AUCo-168 areVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO approximately dose-proportional across the dose range studied. Plasma concentrations of XPAT01-TCE after multiple doses of XPAT01 were detected in 22 participants taking doses of >200 pg / kg XPAT01. Concentrations of XPAT01-TCE ranged from 0.017 to 0.070 pM. On average, maximum XPAT01-TCE concentrations were 0.05% of maximum XPAT01 concentrations. ADA analyses are currently ongoing.Table 4. Preliminary Geometric Mean ( CV) Pharmacokinetic Parameters Following IV Administration of XPAT01 (Part 1: 1-200 pg / kg QW Fixed)Abbreviations: AUCo-ies, area under the concentration-time curve from time 0 to 168 h post-end of infusion;C, Cycle; Cmax, maximum serum concentration; CV, coefficient of variance; D, Day; IV, intravenous; N, number of participants; QW,: once weekly; tmax, time to maximum serum concentration.Notes: Data presented as geometric mean (CV) unless otherwise noted, Individual values presented where N < 2.aData presented as median (range) as time post-end of infusion.bN=3.Table 5. Preliminary Geometric Mean ( CV) Pharmacokinetic Parameters Following IV Administration of XPAT01 (Part 1: 60-1000 pg / kg QW Step-Up)Abbreviations: AUCcues, area under the concentration-time curve from time 0 to 168 h post-end of infusion;C, Cycle; Cmax, maximum serum concentration; CV, coefficient of variance; D, Day; IV, intravenous; QW, once weekly; tmax, time to maximum serum concentration.Notes: N = number of participants. Data presented as geometric mean (CV) unless otherwise noted. Individual values presented where N < 2.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOaData presented as median (range) as time post-end of infusion.bN=27cN=3dN=8Clinical Safety and EfficacySafety

[0213] Preliminary demographics and cancer history are summarized in Table 6. The 60 enrolled and treated participants range in age from 35 to 78 years (median 61 years), with 25.0% aged older than 65 years. Colorectal and breast cancer participants are most frequent at 11 (18.3%) each. For HER2 status, 25 (41.7%) participants had tumors that were IHC3+, 15 (25.0%) participants had tumors that were ISH+, and HER2 mutations were reported in 22 (36.7%) of participant tumors. One participant who was enrolled based on HER2 status eligibility at screening has queries outstanding in the clinical database; HER2 status is missing for this participant pending query resolution.

[0214] Administration of XPAT01 and reasons for treatment discontinuation are summarized in Table 7. Overall, the mean duration of on-treatment follow-up is 72.9 days with a median of 6 administrations of study drug (range: 2-67). Nine of 60 (15.0%) participants treated in escalation Part 1 arc continuing treatment. A total of 25 (41.7%) participants have completed Cycle 2 (i.e., >7 study drug administrations in Table 7). Intraparticipant dose escalations from lower dose cohorts to higher dose cohorts were reported for 8 participants: 1 participant enrolled at 10 pg / kg, 1 at 30 pg / kg, 2 at 60 pg / kg, 1 at 60 / 120 / 120 pg / kg, and 3 at 60 / 120 / 200 Pg / kg.

[0215] Treatment discontinuation has been reported for 51 (85.0%) participants: 46 due to reported Response Evaluation Criteria in Solid Tumors (RECIST) progressive disease, 2 due to AEs, 2 due to symptomatic deterioration, and 1 due to “other.”

[0216] Table 8 provides an overall summary of TEAEs reported in the XPAT01 clinical database for Part 1. Fifty-nine (98.3%) participants had at least 1 TEAE. Twenty-six (43.3%) participants reported TEAEs Grade >3. Forty-four (73.3%) participants reported TEAEs assessed as related to XPAT01, of which 4 (6.7%) participants reported Grade >3 TEAEs that were assessed as related to XPAT01. A single DLT of Grade 3 tumor flare was reported in a participant with lung metastases and Grade 2 pneumonitis who was receiving a fixed dose ofVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO200 pg / kg QW. After this, step-up dosing was initiated, and no further DLTs have been observed up to a maintenance dose of 800 pg / kg weekly. Additionally, 1 patient with peritoneal metastases of gastric adenocarcinoma and recent ramucirumab treatment developed Grade 3 intestinal obstruction followed by life-threatening (Grade 4) jejunal perforation, possibly related to tumor flare and response (evidence of tumor necrosis). In addition to these 2 participants, the other 2 participants reported Grade 3 -related TEAEs of amylase increased and lipase increased. There have been 3 reported Grade 5 TEAEs (i.e., deaths) during the study, all related to disease progression and not related to treatment. Six participants (10.0%) have discontinued treatment and / or study due to TEAEs, none assessed as related to XPAT01.

[0217] Table 9 summarizes the most frequent TEAEs reported in the XPAT01 clinical database for Part 1 through the cutoff date. The most frequent TEAEs (occurring in >12 [(20.0%] participants) included anemia (n=19; 31.7%), CRS (n=18; 30.0%), fatigue (n=18; 30.0%), nausea (n=16; 26.7%), aspartate aminotransferase increased (n=15; 25.0%), pneumonitis (n=15; 25.0%), asthenia (n=14; 23.3%), diarrhea (n=14; 23.3%), pyrexia (n=13; 21.7%), alanine aminotransferase increased (n=12; 20.0%), and pruritus (n=12; 20.0%). The most frequent TEAEs assessed as related to XPAT01 (occurring in >12 [20.0%] participants) include CRS (n=18; 30.0%), pneumonitis (n=15; 25.0%), asthenia (n=12; 20.0%), diarrhea (n=12; 20.0%), and nausea (n=12; 20.0%). The most prevalent important risks of XPAT01 have been low- grade (Grades 1 and 2) CRS and low-grade pneumonitis, occurring in 30.0% and 25.0% of the 60 participants in Part 1, respectively. None of these AEs have been Grade >2 and all events have been easily manageable without requiring steroid premedication. None of these events have led to treatment or study discontinuation.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOTable 6. XPAT01 Demographics and Cancer History - Safety Population (Part 1)Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOAbbreviations: Colorect, colorectal; Endomet, endometriosis; GEJ, gastroesophageal junction; HER2, human epidermal growth factor receptor 2; HNSCC, head and neck squamous cell carcinoma; IHC2+, immunohistochemistry 2 positive; IHC3+, IHC 3 positive; ISH+, in situ hybridization positive; max, maximum; min, minimum; NSCLC, non-small-cell lung cancer.Note: N / n = number of participants. One participant who was enrolled on HER2 status eligibility at screening has queries outstanding in the clinical database; HER2 status is missing for this participant pending query resolution. a Subjects may appear in more than 1 category and hence the sum of number of subjects in all categories may exceed the total number of subjects in the study.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOAbbreviations: AE, adverse event; max, maximum; min, minimum; SD, standard deviation.Notes: N / n = number of participants. Percentages are calculated using the number of participants in safety population.Table 8. XPAT01 Overall Summary of Treatment-Emergent Adverse Events - Safety Population (Part 1)Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOAbbreviations: AESI, adverse event of special interest; CTCAE, Common Terminology Criteria for Adverse Events; DC, discontinuation; DLT, dose-limiting toxicity; TEAE, treatment-emergent adverse event.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WONotes: Table presents numbers and percentages of participants n (%) over number of TEAEs. Percentages are based on the number of participants treated in each group.TEAEs are defined as adverse events that occur on or after the treatment start date or events that are present before the first infusion of XPAT01 and subsequently worsen in severity.aDose modification includes dose interrupted, dose rate reduced, and dose missed.Table 9. XPAT01 Most Frequent Treatment-Emergent Adverse Events - Safety Population (Parti)Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOAbbreviations: PT, Preferred Term; SOC, System Organ Class.Notes: Table presents number and percentages of participants n (%) over number of events. Percentages are based on N. Subjects are counted once within each analysis (Overall, as well as by SOC and PT) with maximum severity grade of the event.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0218] Table 10 summarizes the serious adverse events (SAEs) reported to the safety database reported so far. Overall, 52 SAEs have been reported to the safety database. The only SAE Preferred Terms (PTs) reported more than once were CRS occurring 15 times; pneumonia occurring 3 times; and abdominal pain, diarrhea, disease progression, fatigue, intestinal obstruction, large intestinal obstruction, pneumonitis, and tumor flare occurring twice each.

[0219] Twenty-four SAEs were assessed as related to XPAT01 by the investigator and / or the Sponsor. These include all 15 SAEs of CRS, 2 SAEs of pneumonitis, and 2 SAEs of tumor flare, consistent with the known important potential risks of XPAT01. The remaining 5 related SAEs include single reports of intestinal obstruction and jejunal perforation (also consistent with the known important potential risk of XPAT01), as well as single reports of abdominal pain, diarrhea, and bronchospasm.Table 10. XPAT01 Serious Adverse Events - Safety DatabaseVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOClinical Results Summary

[0220] In Part 1, 60 participants have been treated at escalating doses of XPAT01 ranging from 1 pg / kg to 200 pg / kg as fixed doses and up to 800 pg / kg as maintenance dosing after a step-up regimen. Two additional participants have been treated with XPAT01 60 pg / kg in Part 2 of the study.

[0221] XPAT01 has been generally well tolerated. A single dose-limiting toxicity (DLT) of Grade 3 tumor flare was reported in a participant with lung metastases and Grade 2 pneumonitis who was receiving a fixed dose of 200 pg / kg QW. After this, step-up dosing was initiated, and no further DLTs have been observed up to a maintenance dose of 800 pg / kg weekly. The most prevalent important risks of XPAT01 have been low-grade cytokinerelease syndrome (CRS) and pneumonitis, occurring in 30.0% and 25.0% of the 60 participants in Part 1, respectively. None of these adverse events (AEs) of CRS or pneumonitis have been Grade >2 and all AEs have been easily manageable without requiring steroid premedication. Additionally, 1 patient with peritoneal metastases of gastric adenocarcinoma and recent ramucirumab treatment developed Grade 3 intestinal obstructionVir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO followed by life-threatening jejunal perforation, possibly related to tumor flare and response (evidence of tumor necrosis). Overall, 44 (73.3%) participants in Part 1 reported treatment- emergent AEs (TEAEs) assessed as related to XPAT01, of whom 4 (6.7%) participants reported Grade >3 TEAEs that were assessed as related to XPAT01 . In addition to the related TEAEs of tumor flarc and intestinal obstruction followed by intestinal perforation in the 2 participants described above, the other 2 participants reported Grade 3 related TEAEs of amylase increased and lipase increased. There have been 3 reported Grade 5 AEs (i.e., deaths) during the study, all related to disease progression and not related to treatment.

[0222] Preliminary PK data are available from 54 participants treated with 1 to 800 pg / kg XPAT01 (Part 1). Cmax and area under the concentration time-curve (AUG) from 0 to 168 hours post-end of infusion (AUCo-ies) are approximately dose-proportional across the dose range studied. As of the cutoff date, plasma concentrations of XPAT01-TCE after multiple doses of XPAT01 were detected in 21 participants taking doses of >200 pg / kg XPAT01. Concentrations of XPAT01-TCE ranged from 0.017 to 0.070 pM. On average, maximum XPAT01-TCE concentrations were 0.05% of maximum XPAT01 concentrations.

[0223] The stark contrast between high levels of masked XPAT01 and minimal amounts of unmasked XPAT01-TCE molecules can be seen in the pharmacokinetic data from step-up dosing (FIG. 2A). The step-up dosing regimens included 100 / 200 / 400 pg / kg, 100 / 250 / 600 pg / kg, 100 / 300 / 800 pg / kg, and 100 / 300 / 1000 pg / kg. After each dosing, the plasma concentration of XPAT01 was a few magnitudes higher than that of XPAT01-TCE, underscoring the instant technology’s effectiveness in preventing systemic T-cell engager activation outside the tumor environment, contributing to the tolerable safety profile of XPTA01. Further, FIG. 2A shows a predictable pattern of peaking after each dose and declining linearly.

[0224] XPATOl’s pharmacokinetics after a first dose, across a range of doses, is shown in FIG. 2B, which demonstrates clear linear and dose-proportional relationships. Such linear and dose-proportional relationships warrants predictable dosing and exposure. Along with the observed 5 to 6 days half-life, the data support Q3W (once every three weeks) dosing.Example 3. Follow-up Clinical Evaluation of XPAT01

[0225] This example describes additional data obtained after the cutoff date in Example 2.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0226] In the Part 1 monotherapy dose escalation study, the maximum maintenance dose for weekly dosing was raised to 1000 pg / kg, and the maximum maintenance dose for once every three weeks dosing was 800 pg / kg. A total of 77 patients were enrolled, who were HER2+(IHC2-3+, ISH+, or HER2 mutants). In the Part 2 combo therapy with 200 mg Q3W pembrolizumab, a total of 9 patients were enrolled. XPAT01 was dosed at 60 pg / kg QW or higher.

[0227] At all tested doses, the monotherapy was well tolerated, with Grade 3+ related adverse events of about 8%, most of which related to tumor inflammation. The safety infomiation is summarized in Table 11.Table 11. Best in class safety profile of XPAT01 monotherapy(1) 13 / 21 pneumonitis cases had lung metastases, of the remaining 8, 1 had prior radiation to thorax(2) After the data cut an additional patient after 300 mcg / kg had related grade 3 AE of Pneumonitis; not yet entered as DLT

[0228] It is worth noting that the CRS resulted from the treatments were of low grade, and thus no prophylactic steroid treatment was required to mitigate such adverse effects.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO

[0229] The XPAT01 monotherapy led to significant tumor shrinkage in HER2+ tumor types at doses > 400 pg / kg. Fifty percent (50%) of the patients had observed tumor shrinkage (10 / 20 patients), with a DCR (disease control rate) of 80% (FIG. 3). Responses were observed in many patients with up to 9 prior lines of treatment.

[0230] Quite importantly, the XPAT01 monotherapy was active in heavily pretreated HER2+CRC patients, in particular at doses of 400 pg / kg or higher. As shown in FIG. 4, some CRC patients that had up to 6 lines of prior treatments were responsive to XPAT01. The response is summarized in Table 12. 33% of the CRC patients had a response and 100% showed biomarker responses. Up to 18.1 months duration of response has been observed (patient remains on study).Table 12. Summary of Response among Heavily Pre-Treated Patients

[0231] Various doses of XPAT01 resulted in rapid tumor shrinkage (FIG. 5). CEA responses may precede tumor shrinkage (especially at lower doses) and may be a more sensitive marker of anti-tumor activity. Also importantly, intrapatient dose escalation may deepen responses.

[0232] CEA response preceding tumor shrinkage is illustrated (FIG. 6A-B) in patient 255- 1002 (for up to 800 days of follow-up), who exhibited cPR from HER2+ CRC at week 54 and week 58. This patient was a 57 Year-old male with colorectal cancer (MSS / TMB Low), HER2 status: IHC 3+, prior treatments: 7 prior lines; last treatment: TAS-102; prior Rx with trastuzumab / tucatinib.

[0233] In another patient, who had HER2+breast cancer and underwent 9 lines of prior treatments including T-DXd, 1000 pg / kg of XPAT01 resulted in 52% reduction of SLD (sum of longest diameter) on Cycle 4.

[0234] CRS is a common concern for T-cell engagers. Among prototypical inflammatory mediators elevated in CRS, IL-6 is considered a crucial contributor to systemic toxicides. High circulating IL-6 concentrations causatively contribute to CRS symptoms such as feverVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO and elevation of inflammatory markers, including C-reactive protein (CRP). In patients treated with fixed-dose XPAT01, maximum IL-6 increases were observed after the 1stdose and appeared to attenuate with subsequent doses (FIG. 7). With step-up regimen, maximum IL-6 increases were mostly observed after the 1stdose and appeared to attenuate with subsequent doses (despite the nominal dose increases). Therefore, no prophylactic treatment with steroids is deemed necessary for XPAT01 treatments at the tested doses.* * *

[0235] 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.

[0236] 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

1. Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WOCLAIMSWhat is claimed is:1 . A method for treating cancer in a patient in need thereof, comprising administering to the patient at least 100 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-HER2 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 at least 100 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-HER2 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. The method of claim 1 or 2, wherein the patient is administered 300 pg / kg to 800 pg / kg of the polypeptide once every week to once every four weeks, optionally once every three weeks.

4. The method of claim 1 or 2, wherein the patient is administered 400 pg / kg to 600 pg / kg of the polypeptide once every week to once every four weeks, optionally once every three weeks.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO5. A method for treating cancer in a patient in need thereof, comprising administering to the patient at least 30 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-HER2 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.

6. A method for enhancing immune cell infiltration into a solid tumor in a cancer patient, comprising administering to the patient at least 30 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-HER2 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.

7. The method of claim 5 or 6, wherein the anti-PD- 1 or anti-PD-Ll inhibitor is an anti- PD-1 or anti-PD-Ll antibody.

8. The method of claim 5 or 6, wherein the anti-PD- 1 or anti-PD-Ll inhibitor is pembrolizumab.

9. The method of claim 8, wherein the pembrolizumab is administered at 200 mg once every three weeks, or 400 mg once every six weeks.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO10. The method of any one of claims 5-9, wherein the cancer is characterized with PD-L1 expression.

11. The method of any one of claims 5-10, wherein the patient is administered 40 pg / kg to 500 pg / kg of the polypeptide once every week to once every four weeks, optionally once every three weeks.

12. The method of claim 1 1, wherein the patient is administered 50 pg / kg to 70 pg / kg of the polypeptide once every week to once every four weeks, optionally once every three weeks.

13. The method of any one of claims 1-12, wherein the polypeptide is administered to the patient at a first dose, a second dose later than and at least 40% higher than the first dose, and a third dose later than and at least 40% higher than the second dose.

14. The method of claim 13, wherein the second dose is at least 80% higher than the first dose and the third dose is at least 80% higher than the second dose.

15. The method of claim 13 or 14, wherein the third dose is 2 to 12 weeks after the first dose.

16. The method of any one of claims 1-4 and 13-15, wherein the third dose is 400 pg / kg to 1500 pg / kg.

17. The method of claim 16, wherein the third dose is 600 pg / kg to 1200 pg / kg.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO18. The method of any one of claims 5-15, wherein the third dose is 50 pg / kg to 600 fg / kg-19. The method of claim 18, wherein the third dose is 70 jig / kg to 400 pg / kg.

20. The method of any one of claims 1-19, wherein the administration results in decrease of expression of CEA (carcinoembryonic antigen), CA9-19, ctDNA, or CA-125 in the patient.

21. The method of any one of claims 1-20, wherein the cancer is characterized with HER2 expression.

22. The method of claim 21, wherein the cancer is characterized with HER2 immunohistochemistry (IHC) grade 2+ or 3+, in situ hybridization positive (ISH+), or an activating HER2 mutation.

23. The method of claim 21, wherein the cancer is selected from the group consisting of breast cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, ovarian cancer, urothelial cancer, bladder cancer, colorectal cancer, endometrial cancer, head and neck cancer, lung cancer and salivary gland cancer.

24. The method of claim 22, wherein the cancer patient is resistant to or has progressed from one or more lines of prior treatments.

25. The method of claim 24, wherein the one or more lines of prior treatments comprise an anti-HER2 therapy.Vir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO26. The method of claim 25, wherein the anti-HER2 therapy comprises one or more of trastuzumab, trastuzumab deruxtecan, tucatinib or lapatinib.

27. The method of claim 23, wherein the cancer patient is a breast cancer patient that has undergone or is resistant to at least one line of prior treatments.

28. The method of claim 23, wherein the cancer patient has gastric cancer or gastroesophageal junction cancer and has undergone or is resistant to at least one line of prior treatments.

29. The method of claim 23, wherein the cancer patient has metastatic non-small cell lung cancer (NSCLC) or metastatic colorectal cancer (CRC) and has progressed on a prior treatment.

30. The method of claim 29, wherein the CRC is characterized as microsatellite stable (MSS).

31. The method of any one of claims 1-30, further comprising administering to the patient a steroid.

32. The method of claim 31 , wherein the steroid is administered prior to the first administration of the polypeptide.

33. The method of claim 31 , wherein the steroid is administered after detection of a cytokine release syndrome (CRS) or pneumonitis in the patient.

34. The method of claim 33, wherein detection of the CRS comprises detection of an increase of IL-6 expression in the patient.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO35. The method of any one of claims 31-34, wherein the steroid is dexamethasone, and the dexamethasone is administered for no more than 20 mg every 6 hours or longer.

36. The method of any one of claims 1-30, wherein the patient is not treated with a steroid during administration of the polypeptide or is not pre -treated with a steroid.

37. The method of preceding claim, wherein the anti-HER2 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.

38. The method of 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.

39. The method of preceding claim, wherein the first and second protease-cleavable sites each, independently, has at least 90% sequence identity to SEQ ID NO:3.

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

41. The method of claim 40, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

42. A method for treating cancer in a patient in need thereof, comprising administering to the patient a polypeptide in a dosing regimen comprising:Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO(i) a first dosing cycle (Cl) comprising administering to the patient a target dose of the polypeptide: and(ii) a second dosing cycle (C2) comprising administering to the patient the target dose of the polypeptide; wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

43. The method of claim 42, wherein Cl comprises administering to the patient a first step-up dose of the polypeptide, a second step-up dose of the polypeptide, a target dose of the polypeptide, and optionally a third step-up dose of the polypeptide.

44. The method of claim 43, wherein the second step-up dose is administered to the patient prior to the target dose, and the first step-up dose is administered to the patient prior to the second step-up dose, and wherein the optional third step-up dose is administered to the patient prior to the target dose and after the second step-up dose.

45. The method of claim 43, wherein the first step-up dose is administered to the patient in week 1 of C 1 , the second step-up dose is administered to the patient 6-8 days after the first step-up dose, and the target dose is administered to the patient 6-8 days after the second step- up dose.

46. The method of claim 43, wherein the first step-up dose is administered to the patient on Day 1 of Cl, the second step-up dose is administered to the patient on Day 8 of Cl, and the target dose is administered to the patient on Day 15 of Cl.

47. The method of claim 43, wherein the first step-up dose is administered to the patient in week 1 of Cl, the second step-up dose is administered to the patient 6-8 days after the first step-up dose, the third step-up dose is administered to the patient 6-8 days after the secondVir Ref. No.: S0233.WO1PCT Attorney Docket No: 95KG-401153-WO step-up dose, and the target dose is administered to the patient 6-8 days after the third step-up dose.

48. The method of 43, wherein the first step-up dose is administered to the patient on Day 1 of Cl, the second step-up dose is administered to the patient on Day 8 of Cl, the third step- up dose is administered to the patient on Day 15 of Cl, and the target dose is administered to the patient on Day 22 of C 1.

49. The method of any one of claims 43-48, wherein C2 consists of administering to the patient a single target dose to the patient.

50. The method of any one of claims 43-49, wherein the target dose is administered to the patient on Day 1 of C2.

51. The method of any one of claims 43-50, wherein the target dose is between 300 ug / kg to 1000 ug / kg.

52. The method of claim 51, wherein the target dose is 200 ug / kg, 300 ug / kg, 400 ug / kg, 500 ug / kg, 600 ug / kg, 700 ug / kg, 800 ug / kg, 900 ug / kg or 1000 ug / kg.

53. The method of any one of claims 43-52, wherein the second step-up dose is less than the target dose.

54. The method of any one of claims 43-53, wherein the first step-up dose is less than the second step-up dose.

55. The method of claim any one of claims 43-54, wherein the third step-up dose is less than the target dose and greater than the second step-up dose.Vir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO56. The method of any one of claims 42-55, wherein the dosing regimen further comprises one, two, three, four, five, six, seven, eight, nine, or ten additional dosing cycles.

57. The method of any one of claims 42-56, wherein the polypeptide is administered to the patient as a monotherapy.

58. The method of any one of claims 42-56, wherein the dosing regimen further comprises administering to the patient an additional therapeutic agent.

59. The method of claim 58, wherein the additional therapeutic agent is an anti-PD-1 or anti-PD-Ll inhibitor.

60. The method of claim 5 , wherein the anti-PD-1 or anti-PD-Ll inhibitor is pembrolizumab.

61. The method of claim 60, wherein the pembrolizumab is administered at 200 mg once every three weeks, or 400 mg once every six weeks.

62. The method of any one of claims 42-61, wherein the cancer is characterized with HER2 expression.

63. The method of claim 62, wherein the cancer is characterized with HER2 immunohistochemistry (IHC) grade 2+ or 3+, in situ hybridization positive (ISH+), or an activating HER2 mutation.

64. The method of claim 62 or 63, wherein the cancer is selected from the group consisting of breast cancer, gastric cancer, esophageal cancer, gastroesophageal junctionVir Ref. No.: S0233.WO1PCTAttorney Docket No: 95KG-401153-WO cancer, ovarian cancer, urothelial cancer, bladder cancer, colorectal cancer, endometrial cancer, head and neck cancer, lung cancer and salivary gland cancer.

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