NKG2D FUSION PROTEIN CANCER THERAPY

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application nos. 63/422,893, filed November 4, 2022, 63/508,686, filed June 16, 2023, and 63/517,966, filed August 7, 2023, the contents of each of which are incorporated herein in their entireties by reference thereto.

2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 18, 2023, is named PAT059433-WO-PCT_ST26 SQL.xml and is 89,565 bytes in size.

3. FIELD OF INVENTION

[0003] The disclosure generally relates to NKG2D fusion proteins for use in treating cancer, for example lung cancer, esophageal cancer, renal cancer, and head and neck cancer.

4. BACKGROUND

[0004] Cancer is the second leading cause of death worldwide behind cardiovascular disease (Nagai & Kim, 2017, J Thorac Dis. 9(3): 448-451). Globally, esophageal squamous cell carcinoma is the predominant histological subtype of esophageal cancer, accounting for approximately 90% of the >600,000 cases of esophageal cancerthat occurred in 2020 worldwide (Sung et al., 2021 , CA Cancer J Clin 71 (3):209-249). There were approximately 2.3 million cases of lung cancer in 2020 (Sung et al., 2021 , CA Cancer J Clin 71 (3):209- 249). Non-small cell lung cancer (NSCLC) makes up approximately 85% of lung cancer diagnoses. Renal cell carcinoma accounts for approximately 2% of cancer diagnoses globally, with >400,000 diagnoses and 175,000 deaths in 2018. Clear cell histology is the most common (75% of RCC) subtype. Head and neck squamous cell carcinoma (HNSCC) is a common cancer worldwide, with rising incidence, including nearly 900,000 cases and 450,000 deaths in 2018, with over a million cases expected by 2030. HPV-associated HNSCC comprises the majority of patients with oropharyngeal HNSCC (approximately 70%). While the use of immune checkpoint inhibitors has resulted in survival improvements in all four indications, additional therapeutics, for example for patients progressing after currently available/approved treatments, are needed. Five-year survival for patients with metastatic esophageal, NSCLC, and RCC all remain low, ranging from 5% for esophageal cancer, 7% for non-small cell lung cancer, and 13% for renal cell carcinoma (American Cancer Society, 2019, Cancer A-Z, cancer.org/cancer.html). Metastatic oral cavity/oropharyngeal HNSCC (including HPV positive and HPV negative) has a five-year survival of only 32%. [0005] Accordingly, there is a need for new cancer treatments, including treatments for lung cancer, esophageal cancer, renal cancer, and head and neck cancer.

5. SUMMARY

[0006] This disclosure relates to treatment of cancers, such lung cancer, esophageal cancer, renal cancer, head and neck cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, breast cancer, prostate cancer, stomach cancer, bladder cancer, hepatocellular carcinoma (HCC), nasopharyngeal carcinoma (NPC), melanoma, plasma cell cancer, bone cancer, soft tissue cancer, glioblastoma multiforme, astrocytoma, or hematological cancer, with NKG2D fusion proteins (e.g., the NKG2D fusion protein identified herein as “HH8”).

[0007] Accordingly, in one aspect, the disclosure provides methods of treating a subject having a cancer comprising administering to the subject a therapeutically effective amount of an NKG2D fusion protein (e.g., HH8).

[0008] In another aspect, the disclosure provides NKG2D fusion proteins (e.g., HH8) for use in a method of treating a subject having a cancer.

[0009] NKG2D fusion proteins of the disclosure are typically dimeric proteins comprising two monomers, each monomer comprising a first NKG2D peptide or variant thereof, a second NKG2D peptide or variant thereof, a first peptide linker connecting said first NKG2D peptide or variant thereof and said second NKG2D peptide or variant thereof, and a fragment crystallizable region (Fc region) of an immunoglobulin (Ig). Without being bound by theory, it is believed that such NKG2D fusion proteins may act by one or more of the following mechanisms: (1) promotion of ADCC activity by binding to NKG2D-L expressed on the surface of tumor cells, leading to crosslinking of Fc receptors expressed on immune cells and induction of cancer cell killing; (2) neutralization of tumor cell surface NKG2D-L and prevention of downregulation of NKG2D; and (3) neutralization of soluble NKG2D-L derived from tumor cell shedding to prevent the downregulation of NKG2D and systemic immune desensitization. As such, it is believed that the NKG2D fusion proteins can promote direct killing of tumor cells and indirect recovery of immune function by preventing NKG2D-based desensitization. Amino acid sequences of exemplary monomers that can be included in dimeric NKG2D fusion proteins are set forth in SEQ ID NOs: 17-40. In some embodiments, the NKG2D fusion protein is a dimer of monomers each having an amino acid sequence as set forth in SEQ ID NO:23.

[0010] In some embodiments of the methods and compositions of the disclosure, the subject has an advanced or metastatic cancer. Exemplary cancers that can treated by the methods of the disclosure and with the compositions of the disclosure include lung cancer, esophageal cancer, renal cancer, head and neck cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, breast cancer, prostate cancer, stomach cancer, bladder cancer, hepatocellular carcinoma (HCC), nasopharyngeal carcinoma (NPC), melanoma, plasma cell cancer, bone cancer, soft tissue cancer, glioblastoma multiforme, astrocytoma, and hematological cancers.

[0011] In some embodiments, the cancer is lung cancer (e.g., non-squamous lung cancer, squamous lung cancer, or non-small cell lung cancer (e.g., of squamous or non-squamous histology)), esophageal cancer (e.g., esophageal squamous cell carcinoma), renal cancer (e.g., renal cell carcinoma, for example of clear cell histology), or a head and neck cancer (e.g., head and neck squamous cell carcinoma and/or HPV-associated head and neck cancer).

[0012] In another aspect, the disclosure provides dosing regimens for NKG2D fusion proteins that can be used in methods of treating cancer, for example any of the cancers described herein.

[0013] Exemplary NKG2D fusion proteins are described in Section 7.2 and specific embodiments 1 , 2 and 151 to 178, infra.

[0014] Exemplary cancers that can be treated with NKG2D fusion proteins and exemplary subject populations are described in Section 7.3 and specific embodiments 79 to 118 and 183 to 186, infra.

[0015] Exemplary dosing regimens and administration regimens for NKG2D fusion proteins that can be used in methods of treating cancer are described in Section 7.4 and specific embodiments 3 to 78, 119 to 150, and 179 to 182, infra.

6. BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1 shows a schematic of the PK/PD model used to determine human doses of HH8 (Example 1).

[0017] FIGS. 2A-2C show simulated free concentration of HH8 in serum for i.v. doses of 70 mg, 210 mg, 700 mg, and 1400 mg Q2W for a 70 kg subject (FIG. 2A); the minimum free concentration of HH8 over the 2-week dosing interval, Cmin, as a function of dose with colored bars highlighted to represent the doses shown in FIG. 2A (FIG. 2B); the minimum free concentration of HH8 over the 2-week dosing interval, Cmin, within the tumor as a function of dose (FIG. 2C) (Example 1). [0018] FIGS. 3AA-3CC show predicted free HH8 PK and derived Cmin in serum and tumor with Q2W (FIG. 3AA-3AC), Q3W (FIG. 3BA-3BC), and Q4W (FIG. 3CA-3CC) dosing in units of pg/ml (Example 1).

[0019] FIG. 4 shows predicted intra-tumor MICA neutralization (Example 1).

[0020] FIGS. 5A-5B show predicted human serum MICA neutralization (FIG. 5A) and minimum serum soluble MICA neutralization (FIG. 5B) (Example 1).

[0021] FIGS. 6A-6C show model predictions by mechanism of action (Example 1). FIG. 6A: intratumoral ADCC; FIG. 6B: tumor cell membrane ligand neutralization; FIG. 6C: systemic soluble ligand neutralization. Shading shows model prediction trend. Predictions were based on Cmin during dosing interval.

[0022] FIGS. 7A-7D show HH8 model predictions by MoA with experimental context (Example 1). FIG. 7A: intratumoral ADCC; FIG. 7B: tumor cell membrane ligand neutralization; FIG. 7C: systemic soluble ligand neutralization; FIG. 7D: legend for FIGS. 7A- 7C.

[0023] FIGS. 8A-8D show a comparison between MoA prediction for Q2W versus Q3W dosing (Example 1). FIG. 8A: intratumoral ADCC; FIG. 8B: tumor cell membrane ligand neutralization; FIG. 8C: systemic soluble ligand neutralization; FIG. 8D: legend for FIGS. 8A- 8C.

[0024] FIGS. 9A-D show predicted MoA engagement at starting dose of 70 mg Q2W based on Cavg simulations (Example 1). FIG. 9A: intratumoral ADCC; FIG. 9B: tumor cell membrane ligand neutralization; FIG. 9C: systemic soluble ligand neutralization; FIG. 9D: legend for FIGS. 9A-9C. 70 mg dose is indicated by vertical dashed line.

[0025] FIGS. 10A-10C shows predicted time above threshold for MoAs (Example 1). FIG. 10A: intratumoral ADCC; FIG. 10B: tumor cell membrane ligand neutralization; FIG. 10C: systemic soluble ligand neutralization. In FIGS. 10A-10B, shaded regions indicate model prediction at 90% Cl for each shown metric. In FIG. 10C, shaded regions indicate model prediction at 90% Cl and 97% Cl, as indicated in figure.

[0026] FIG. 11 shows the study design of Example 2.

7. DETAILED DESCRIPTION

[0027] In one aspect, the disclosure provides methods of treating a subject having a cancer comprising administering to the subject a therapeutically effective amount of an NKG2D fusion protein. [0028] In another aspect, the disclosure provides NKG2D fusion proteins for use in a method of treating a subject having a cancer.

[0029] In another aspect, the disclosure provides dosing regimens for NKG2D fusion proteins that can be used in methods of treating cancer, for example lung cancer, esophageal cancer, renal cancer, and head and neck cancer.

[0030] Exemplary NKG2D fusion proteins are described in Section 7.2 and specific embodiments 1 , 2 and 151 to 178, infra.

[0031] Exemplary cancers that can be treated with NKG2D fusion proteins and exemplary subject populations are described in Section 7.3 and specific embodiments 79 to 118 and 183 to 186, infra.

[0032] Exemplary dosing regimens and administration regimens for NKG2D fusion proteins that can be used in methods of treating cancer are described in Section 7.4 and specific embodiments 3 to 78, 119 to 150, and 179 to 182, infra.

7.1. Definitions

[0033] As used herein, the following terms are intended to have the following meanings:

[0034] A, An, The: As used herein, the term "a", "an", "the" and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0035] And/or: The term "and/or" means that each one or both or all the components or features of a list are possible variants, especially two or more thereof in an alternative or cumulative way.

[0036] Or: Unless indicated otherwise, an “or” conjunction is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in the alternative (A or B, where the selection of A is mutually exclusive from B) and the selection of features in conjunction (A or B, where both A and B are selected). In some places in the text, the term “and/or” is used for the same purpose, which shall not be construed to imply that “or” is used with reference to mutually exclusive alternatives.

[0037] Advanced cancer: The term “advanced cancer” describes a cancer that has spread from the original site and/or has recurred and/or is unlikely to be cured. In some embodiments, an advanced cancer is a cancer that has reached at least Stage HA, at least Stage IIB, at least Stage IIIA, or at least Stage IIIB. In some embodiments, an advanced cancer is a locally advanced cancer. In other embodiments, an advanced cancer is a metastatic cancer (e.g., Stage IVA or Stage IVB).

[0038] Anti-PD-1 agent and Anti-PD-L1 agent: The terms “anti-PD-1 agent” and anti-PD- L1 agent” as used herein refers to agents that are used to block the activity of PD-1 and PD- L1 , respectively. Anti-PD-1 and anti-PD-L1 agents include anti-PD-1 and anti-PD-L1 antibodies, for example, Tislelizumab, Nivolumab, Pembrolizumab, Cemiplimab and Dostarlimab (anti-PD-1 agents); and Atezolizumab, Avelumab, and Durvalumab (anti-PD-L1 agents).

[0039] Anti-TGF-B agent: The terms “anti-TGF-|3 agent” as used herein refers to agents that are used to block the activity of one or more TGF-|3 isoforms. Anti-TGF-p agents include anti-TGF-|3 antibodies, for example, NIS793 and Fresolimumab.

[0040] Cancer: The term “cancer” refers to a disease characterized by the uncontrolled (and often rapid) growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of cancers include the lung, esophageal, renal, head and neck, pancreatic, gastric, colorectal, ovarian, endometrial, biliary tract, liver, breast, prostate, bladder, hepatocellular carcinoma (HCC), nasopharyngeal carcinoma (NPC), melanoma, plasma cell, bone, soft tissue, glioblastoma multiforme, astrocytoma, and hematological cancers described herein. Solid cancers can be classified according to the AJCC Cancer Staging Manual, Eighth Edition (Amin et al., eds.). References herein to a cancer stage, for example, Stage 11 IB, Stage IIIC, and Stage IV, refer to cancer stages according to the AJCC Cancer Staging Manual, Eighth Edition.

[0041] Chemotherapeutic agent: The term “chemotherapeutic agent” or “chemotherapy agent” as used herein refers to an agent that is used to directly or indirectly inhibit the uncontrolled proliferation of cancer cells. Examples of chemotherapeutic agents include microtubule stabilizing agents such as paclitaxel and nab-paclitaxel, nucleoside analogs such as gemcitabine, platinum-based agents such as carboplatin and cisplatin, and antifolates such as pemetrexed. Platinum-based agents contain a platinum atom, while non- platinum-based agents do not contain a platinum atom.

[0042] Combination: The term “combination” refers to a set of two or more individual therapeutic agents. The terms “a combination” or “in combination with” is not intended to imply that the therapy or the therapeutic agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope described herein. The therapeutic agents in the combination can be administered concurrently with, prior to, or after, one or more other additional therapies or therapeutic agents. The therapeutic agents or therapeutic protocol can be administered in any order. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. It will further be appreciated that the additional therapeutic agent utilized in a combination may be administered together or separately in different compositions. In general, it is expected that additional therapeutic agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

[0043] ECOG performance status score: The term “ECOG performance status score” as used herein refers to a subject’s score on the Eastern Cooperative Oncology Group (ECOG) performance status score, as described in Oken et al., 1982, Am J Clin Oncol 5(6):649-55.

Scores can range from 0 to 5:

[0044] Effective amount: By the term "effective amount" or "therapeutically effective amount" or "pharmaceutically effective amount", is meant the amount or quantity of active agent (or combination of agents) that is sufficient to elicit the required or desired response, or in other words, the amount that is sufficient to elicit an appreciable biological response when administered to a subject. Said amount preferably relates to an amount that is therapeutically or in a broader sense also prophylactically effective against the progression of a disease or disorder as disclosed herein. It is understood that an “effective amount" or a “therapeutically effective amount" can vary from subject to subject, due to variation in metabolism of an agent, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. [0045] Fragment crystallizable region (Fc region): The term “fragment crystallizable” or “Fc region” as used herein is meant the polypeptide comprising the CH2-CH3 domains of an IgG molecule, and in some cases, inclusive of the hinge. In EU numbering for human IgG 1 , the CH2-CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230. Thus the definition of “Fc region” includes both (CH2-CH3) or (hinge-CH2-CH3), or fragments thereof. An “Fc fragment” in this context can contain fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another Fc region as can be detected using standard methods, generally based on size (e.g., non-denaturing chromatography, size exclusion chromatography).

[0046] Locally advanced cancer: The term “locally advanced cancer” describes a cancer that has spread from the original site to nearby tissues and/or nearby lymph nodes. In some embodiments, a locally advanced cancer is a cancer that has reached at least Stage 11 B, at least Stage 11 IA, or at least Stage 11 IB, but has not reached Stage IV.

[0047] Metastatic cancer: The term “metastatic cancer” describes a cancer that has recognizable origins in one part of the body (e.g., lung) and has spread to other parts of the body. For example, a metastatic lung cancer is a cancer having origin in the lung and which has spread to another part(s) of the body, for example distant lymph nodes, bones, brain, liver, or adrenal glands. Metastatic cancers include Stage IVA and Stage IVB cancers.

[0048] Monotherapy: As used herein, “monotherapy” refers to the administration of a single active or therapeutic compound to a subject in need thereof. Preferably, monotherapy will involve administration of a therapeutically effective amount of an active composition (e.g., an NKG2D fusion protein, or any composition described herein). For example, described herein can be a cancer monotherapy with one of compositions described herein administered to a subject in need of for treatment of cancer. Monotherapy may be contrasted with combination therapy, in which a combination of multiple active compositions are administered.

[0049] NKG2D: NKG2D, also referred to as KLRK1 ; killer cell lectin-like receptor subfamily K, member 1 ; CD314; KLR; NKG2-D; FLJ17759; FLJ75772 or D12S2489E, a transmembrane protein belonging to the CD94/NKG2 family of C-type lectin-like receptors. In humans, it is expressed by NK cells, y6 T cells and CD8+ a T cells. NKG2D recognizes induced-self proteins from MIC and RAET1/ULBP families which appear on the surface of stressed, malignant transformed, and infected cells. NKG2D described herein includes any of NKG2D’s naturally occurring forms, or variants that maintain its protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native NKG2D). In some embodiments, NKG2D variants or homologues have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring NKG2D. In other embodiments, NKG2D is the protein as identified by its NCBI sequence reference NP_031386. In other embodiments, NKG2D is substantially identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the protein as identified by its NCBI sequence reference (NP_031386) or functional fragment thereof.

[0050] NKG2D fusion protein: The term “NKG2D fusion protein” refers to a protein comprising a first NKG2D peptide or variant thereof, a second NKG2D peptide or variant thereof, a peptide linker connecting the first NKG2D peptide or variant thereof and the second NKG2D peptide or variant thereof, and a fragment crystallizable region (Fc region) of an immunoglobulin (Ig). NKG2D fusion proteins are typically dimeric, comprising two monomers, each comprising a first NKG2D peptide or variant thereof, a second NKG2D peptide or variant thereof, a peptide linker connecting the first NKG2D peptide or variant thereof and the second NKG2D peptide or variant thereof, and a fragment crystallizable region (Fc region) of an immunoglobulin (Ig). Dimeric NKG2D fusion proteins can be homodimers or heterodimers.

[0051] Patient/subject: As used herein, the term "patient" or "subject" are taken to mean a human. Except when noted, the terms “patient” or “subject” are used herein interchangeably.

[0052] PD1 : The term “PD1” refers to Programmed cell death protein 1 . The human amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, an amino acid sequence of human PD1 can be found as UniProt Accession No. Q15116.

[0053] PD-L1 : The term “PD-L1” refers to Programmed death-ligand 1. The human amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, an amino acid sequence of human PD-L1 can be found as UniProt Accession No. Q9NZQ7.

[0054] Polypeptide: The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0055] Sequence identity: As used herein, the percentage sequence identity between two amino acid sequences is calculated by multiplying the number of matches between a pair of aligned sequences by 100 and dividing by the length of the aligned region. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another, nor does it consider substitutions or deletions as matches. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, by manual alignment or using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for achieving maximum alignment.

[0056] TGF-B: The term “TGF-p” refers to transforming growth factor beta. The transforming growth factor beta (TGF-P) protein family consists of three distinct isoforms found in mammals (TGFpi , TGFpi , and TGFpi). The human amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, amino acid sequences of human TGFpi , TGFpi , and TGFpi proproteins can be found as UniProt Accession Nos. P01137, P61812, and P10600, respectively.

[0057] Treat, treating, treatment: As used herein, the term “treat”, “treating" or "treatment" of any disease or disorder refers in one embodiment to ameliorating the disease or disorder (e.g., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms or pathological features thereof). In another embodiment “treat”, "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter or pathological features of the disease, e.g., including those, which may not be discernible by the subject. In yet another embodiment, “treat”, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of at least one discernible or non-discernible symptom), physiologically (e.g., stabilization of a physical parameter) or both. In yet another embodiment, “treat”, "treating" or "treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder, or of at least one symptoms or pathological features associated thereof. In yet another embodiment, “treat”, "treating" or "treatment" refers to preventing or delaying progression of the disease to a more advanced stage or a more serious condition. The benefit to a patient to be treated is either statistically significant or at least perceptible to the patient or to the physician. However, it will be appreciated that when a medicament is administered to a patient to treat a disease, the outcome may not always be an effective treatment.

[0058] Tumor: The term “tumor” is used interchangeably with the term “cancer” herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors.

[0059] The following abbreviations are used throughout the disclosure:

7.2. NKG2D Fusion Proteins

[0060] NKG2D fusion proteins are known, for example, from WO 2021/053556, WO 2010/080124, and WO 2017/083612, the contents of which are incorporated herein by reference in their entireties.

[0061] In some embodiments, an NKG2D fusion protein used in the methods and compositions of the disclosure is a dimeric protein comprising two monomers, each comprising a first NKG2D peptide or variant thereof, a second NKG2D peptide or variant thereof, a first peptide linker connecting the first NKG2D peptide or variant thereof and the second NKG2D peptide or variant thereof, and a fragment crystallizable region (Fc region) of an immunoglobulin (Ig). The two monomers can be the same or different. In some embodiments, the monomers are the same. In other embodiments, the monomers are different. Likewise, the first NKG2D peptide and second NKG2D peptide can be the same or different. In some embodiments, the first NKG2D peptide and second NKG2D peptide are the same. In other embodiments, the first NKG2D peptide and second NKG2D peptide are different.

[0062] Exemplary NKG2D peptides as set forth in Table 1 .

[0063] In some embodiments, the first NKG2D peptide comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1 . In other embodiments, the first NKG2D peptide comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2. In other embodiments, the first NKG2D peptide comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:3. In other embodiments, the first NKG2D peptide comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:4. In some embodiments, the first NKG2D peptide comprises an amino acid sequence of SEQ ID NO:1. In other embodiments, the first NKG2D peptide comprises an amino acid sequence of SEQ ID NO:2. In other embodiments, the first NKG2D peptide comprises an amino acid sequence of SEQ ID NO:3. In other embodiments, the first NKG2D peptide comprises an amino acid sequence of SEQ ID NO:4. [0064] In some embodiments, the second NKG2D peptide comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1. In other embodiments, the second NKG2D peptide comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2. In other embodiments, the second NKG2D peptide comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:3. In other embodiments, the second NKG2D peptide comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:4. In some embodiments, the second NKG2D peptide comprises an amino acid sequence of SEQ ID NO:1. In other embodiments, the second NKG2D peptide comprises an amino acid sequence of SEQ ID NO:2. In other embodiments, the second NKG2D peptide comprises an amino acid sequence of SEQ ID NO:3. In other embodiments, the second NKG2D peptide comprises an amino acid sequence of SEQ ID NO:4.

[0065] Exemplary peptide linkers that can be used to link the first NKG2D peptide and the second NKG2D peptide include glycine-serine peptide linkers, glycine-alanine peptide linkers, and glycine-proline peptide linkers. In some embodiments, a glycine-serine linker is represented by the formula (GS) _n , wherein n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 (SEQ ID NO:5). In some embodiments, a peptide linker is a G4S linker represented by the formula of (GGGGS)n, where n is 1, 2, 3, 4, or 5 (SEQ ID NO:6). In some embodiments, a peptide linker is a G ₄ A linker represented by the formula of (GGGGA) _n , where n is 1 , 2, 3, 4, or 5 (SEQ ID NO:7). In some embodiments, a peptide linker is a G ₄ P linker represented by the formula of (GGGGP) _n , where n is 1 , 2, 3, 4, or 5 (SEQ ID NO:8). In some embodiments, a peptide linker is a G ₃ A linker represented by the formula of (GGGA) _n , where n is 1 , 2, 3, 4, or 5 (SEQ ID NO:9). In some embodiments, a peptide linker is represented by the formula of (GGGs)n, where n is 1 , 2, 3, 4, or 5 (SEQ ID NO:10). In some embodiments, the glycine- serine linker is represented by the formula GGGSGGGS (SEQ ID NO: 11).

[0066] The Fc region can be, for example, an Fc region of human immunoglobulin G (IgG). In some embodiments, the Fc region comprises an Fc region of human IgG 1. In some embodiments, the Fc region comprises an Fc region of human lgG2. In some embodiments, the Fc region comprises an Fc region of human lgG3. In some embodiments, the Fc region comprises an Fc region of human lgG4. In some embodiments, the Fc region comprises an Fc region of mouse lgG2a. In some embodiments, the Fc region comprises an Fc region of human IgM. In some embodiments, the Fc region comprises an Fc region of human lgA1 . In some embodiments, the Fc region comprises an Fc region of human lgA2. In some embodiments, the Fc region comprises an Fc region of human IgD. In some embodiments, the Fc region comprises an Fc region of human IgE.

[0067] Exemplary Fc region sequences are set forth in Table 2. [0068] In some embodiments, the Fc region comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:12. In other embodiments, the Fc region comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:13. In other embodiments, the Fc region comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:14. In other embodiments, the Fc region comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 15. In other embodiments, the Fc region comprises an amino acid sequence having at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:16. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:12. In other embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:13. In other embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:14. In other embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:15. In other embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 16.

[0069] Each monomer can comprise, from N-terminus to C-terminus, the first NKG2D peptide or variant thereof, the first peptide linker, the second NKG2D peptide or variant thereof and the Fc region. The Fc region can be directly fused with the second NKGD2 peptide or variant thereof, or can be fused via a peptide linker, for example a peptide linker described in this Section. Alternatively, each monomer can comprise from N-terminus to C- terminus, the Fc region, the first NKG2D peptide or variant thereof, the first peptide linker, and the second NKG2D peptide or variant thereof. The Fc region can be directly fused with the first NKGD2 peptide or variant thereof, or can be fused via a peptide linker, for example a peptide linker described in this Section.

[0070] Exemplary monomer sequences are set forth in Table 3.

[0071] In some embodiments, each monomer comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 49, and 40. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO: 17. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:18. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO: 19. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:20. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:21 . In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:22. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:23. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:24. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:25. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:26. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:27. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:28. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:29. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NQ:30. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:31 . In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:32. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:33. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:34. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:35. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:36. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:37. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:38. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NO:39. In other embodiments, each monomer comprises the amino acid sequence of SEQ ID NQ:40.

[0072] The monomers of a dimeric protein NKG2D fusion protein can be covalently linked, for example by one or more cysteine-cysteine bridges.

7.3. Subject Populations

[0073] An NKG2D fusion proteins of the disclosure can be administered to a subject having cancer, for example an NKG2D ligand expressing cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the subject has a liquid tumor.

[0074] In some embodiments, the cancer is a lung cancer, an esophageal cancer, a renal cancer, a head and neck cancer, a pancreatic cancer, a gastric cancer, a colorectal cancer, an ovarian cancer, an endometrial cancer, a biliary tract cancer, a liver cancer, a breast cancer, a prostate cancer, a stomach cancer, a bladder cancer, a hepatocellular carcinoma (HCC), a nasopharyngeal carcinoma (NPC), a melanoma, a plasma cell cancer, a bone cancer, a soft tissue cancer, a glioblastoma multiforme, an astrocytoma, or a hematological cancer (e.g., a lymphoma, such as Hodgkin’s lymphoma or non-Hodgkin’s lymphoma; a leukemia, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML) or chronic lymphocytic leukemia (CLL); or a myeloma, such as multiple myeloma).

[0075] In some embodiments, the cancer is lung cancer, an esophageal cancer, a renal cancer, or a head and neck cancer.

[0076] In some embodiments, the cancer is lung cancer, for example non-small cell lung cancer (NSCLC). The cancer can be squamous lung cancer. Alternatively, the cancer can be non-squamous lung cancer. In some embodiments, cancer does not have a known activating mutation, for example in EGFR, ALK, ROS1, or RET. Activating mutations include large-scale alterations, such as gain/amplification, insertion, or chromosome translocation, as well as small-scale mutations, such as point mutations (Tuna and Amos, 2012, DOI: 10.5772/48701) Targeted therapies are often used to treat cancers having activating mutations.

[0077] In some embodiments, the cancer is esophageal cancer, for example esophageal squamous cell carcinoma.

[0078] In other embodiments, the cancer is renal cancer, for example renal cell carcinoma. In particular embodiments, the renal cell carcinoma is clear cell renal cell carcinoma.

[0079] In other embodiments, the cancer is head and neck cancer, for example head and neck squamous cell carcinoma (HNSCC). Head and neck cancers include oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, nasopharyngeal cancer, paranasai sinus and nasal cavity cancer, salivary gland cancer, and squamous cell neck cancer. In some embodiments, the head and neck cancer is one of the foregoing types. Head and neck cancer is often associated with human papilloma virus (HPV) infection. Accordingly, in some embodiments the head and neck cancer is HPV- associated head and neck cancer. In some embodiments, the cancer is HPV-associated HNSCC, for example HPV-associated oropharyngeal HNSCC. p16 positivity is highly correlated with HPV-associated head and neck cancer (Stephen et al., 2013, Cancer Clin Oncol. 2(1 ):51 -61 . Accordingly, in some embodiments, the cancer is p16 positive.

[0080] Without being bound by theory, it is believed that subjects having immune-inflamed tumors may respond particularly well to treatment with NKG2D fusion proteins of the disclosure in view of the mechanisms of action outlined in Section 5. Thus, in some embodiments, the subject has a tumor with an immune-inflamed phenotype. PD-L1 is a biomarker whose expression is associated with an immune-inflamed phenotype (Chen & Mellman, 2017, Nature 541 (7637):321-330). Thus, PD-L1 expression can be used to identify subject having immune-inflamed tumors. PD-L1 expression can be measured by an immunohistochemistry (IHC) assay, for example the VENTANA® PD-L1 (SP263) IHC assay (Roche). In some embodiments, a subject treated according to method of the disclosure has PD-L1 expressing cells (e.g., as measured by an IHC assay), for example in > 1% of tumor cells. PD-L1 expression can be assessed, for example, using a tumor biopsy sample. In other embodiments, the cancer is PD-L1 negative (e.g., having PD-L1 expression in <1% of tumor cells).

[0081] The subject’s cancer can be any stage, e.g., Stage I, Stage II, Stage III or Stage IV. For example, the subject can have advanced or metastatic cancer. In some embodiments, the cancer is advanced. In some embodiments, the cancer is locally advanced. In other embodiments, the cancer is metastatic cancer.

[0082] The subject can be a subject who, in the opinion of a clinician, has a cancer that is unlikely to be cured by surgery and/or radiotherapy.

[0083] The subject can have one or more (e.g., at least one or more than one) measurable lesion as defined by RECIST 1.1 criteria prior to administration of the NKG2D fusion protein. RECIST 1.1 criteria are described in Eisenhauer et al. 2009, European Journal of Cancer, 45(2):228-247 doi:10.1016/j.ejca.2008.10.026, the contents of which are incorporated herein by reference in their entireties.

[0084] The subject can be a subject who is receiving and/or has previously received treatment for the cancer prior to treatment with the NKG2D fusion protein, for example treatment with one or more standard of care therapies. For example, a subject having lung cancer can be a subject who is being treated with and/or has been treated with (i) an anti- PD-1 and/or anti-PD-L1 agent and (ii) a platinum-based chemotherapy agent, in combination or in sequence. As another example, a subject having esophageal cancer can be a subject who is being treated with and/or has been treated with (i) an anti-PD-1 and/or anti-PD-L1 agent and (ii) a platinum-based chemotherapy agent, in combination or in sequence. As another example, a subject with renal cancer, e.g., renal cell carcinoma, can be a subject who is being treated with and/or has been treated with (i) an anti-PD-1 and/or anti-PD-L1 agent and (ii) a VEGFR tyrosine kinase inhibitor, in combination or in sequence. As yet another example, a subject with head and neck cancer can be a subject who is being treated with and/or has been treated with (i) an anti-PD-1 and/or anti-PD-L1 agent and (ii) a platinum-based chemotherapy agent, in combination or in sequence.

[0085] The subject can be a subject who is receiving or who has received a standard of care therapy but is not receiving a clinical benefit from the standard of care therapy, for example as assessed by a treating clinician. In some embodiments, the subject is intolerant of or ineligible for a standard of care therapy.

[0086] Treatment of a subject can be continued, for example by periodically administering the NKG2D fusion protein, until the subject achieves remission or disease progression, for example as measured by RECIST 1.1 criteria. In some embodiments, a subject continues to receive treatment with the NKG2D fusion protein until clinically significant disease progression, for example as measured by an increase in ECOG performance status score. In some embodiments, the subject has an ECOG performance status score of 0 or 1 before treatment. The methods of the disclosure may delay disease progression, thereby prolonging progression free-survival.

[0087] In some embodiments, the subject is an adult subject, for example a male or female at least 18 years of age.

[0088] The subject can be a subject meeting one, two, three, four, five, six, seven, or all eight of the inclusion criteria set forth in Section 8.2.2.1. In some embodiments, the subject does not meet any of the exclusion criteria set forth in Section 8.2.2.2.

7.4. NKG2D Fusion Protein Dosing Regimens

[0089] In some aspects, the disclosure provides NKG2D fusion protein dosing regimens.

The dosing regimens can be used in the methods of the disclosure. In some embodiments, a NKG2D fusion protein, for example a dimeric protein as described in Section 7.2, is administered at a dose ranging from 20 mg to 5000 mg, 20 mg to 2500 mg, 35 mg to 4200 mg or 35 mg to 2100mg. Narrower dose ranges can also be used, for example, 35 mg to 70 mg, 35 mg to 210 mg, 35 mg to 700 mg, 35 mg to 2100 mg, 70 mg to 210 mg, 70 mg to 700 mg, 70 mg to 2100 mg, 210 mg to 700 mg, 210 mg to 2100 mg, 700 mg to 2100 mg, or 2100 mg to 4200 mg. Exemplary doses that can be used include 35 mg, 70 mg, 210 mg, 700 mg, 1400 mg, 2100 mg, and 4200 mg doses.

[0090] In some aspects, doses that can be used include 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg,

340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg,

550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg,

760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, 2500 mg, 2600 mg, 2700 mg, 2800 mg, 2900 mg, 3000 mg, 3100 mg, 3200 mg, 3300 mg, 3400 mg, 3500 mg, 3600 mg, 3700 mg, 3800 mg, 3900 mg, 4000 mg, 4100 mg, and 4200 mg. In some embodiments, the dose is 35 mg. In some embodiments, the dose is 70 mg. In some embodiments, the dose is 210 mg. In some embodiments, the dose is 700 mg. In some embodiments, the dose is 1400 mg. In some embodiments, the dose is 2100 mg. In some embodiments, the dose is 4200 mg.

[0082] The dosing regimens of the disclosure typically comprise administering the NKG2D fusion protein periodically, for example once every two weeks, once every three weeks, or once every four weeks. In practice, an administration window can be provided, for example, to accommodate slight variations to a dosing schedule. For example, a window of ± 3 days, ± 2 days, or ± 1 day around the dosage date can be used.

[0092] In some embodiments, the NKG2D fusion protein is administered once every two weeks, for example on day 1 (± 3 days) and day 15 (± 3 days) of a 28-day cycle.

[0093] In other embodiments, the NKG2D fusion protein is administered once every three weeks, for example on day 1 (± 3 days) of a 21 -day cycle.

[0094] In other embodiments, the NKG2D fusion protein is administered once every four weeks, for example on day 1 (± 3 days) of a 28-day cycle.

[0095] A treatment regimen can comprise one cycle or multiple cycles (e.g., multiple 21-day cycles when the NKG2D fusion protein is administered once every three weeks, or multiple 28-day cycles when the fusion protein is administered once every two weeks, once every three weeks, or once every four weeks). For example, a treatment regimen can comprise two or more, three or more, or four or more cycles (e.g., four or more, five or more, or six or more cycles). In some embodiments, a treatment regimen comprises at least six cycles (e.g., six or more, eight or more, 10 or more, 12 or more, 14 or more, etc.) and/or up to about 24 cycles. In some embodiments, a treatment regimen can comprise repeating a cycle until disease progression.

[0096] The same dose of the NKG2D fusion protein can be administered at each administration or, alternatively, a first dose amount can be followed by a second dose amount. For example, if a subject experiences side effects at a first dose amount, the second dose can be a lower dose than the first dose amount. Alternatively, if a first dose amount is well tolerated, the second dose amount can be a higher dose than the first dose amount. A subject’s dose amount can be changed once or more than once, e.g., a third dose amount can be administered following administration of a second dose amount.

[0097] In some embodiments, the first dose amount and second dose amount are both in the range of 20 mg to 5000 mg, 20 mg to 2500 mg, 35 mg to 4200 mg, or 35 mg to 2100 mg. For example, for a first dose amount that is lower than the second dose amount, the first dose amount can in some embodiments range from 35 mg to 2100 mg (e.g., 35 mg, 70 mg, 210 mg, 700 mg or 2100 mg) and the second dose amount can range from 70 mg to 4200 mg (e.g., 70 mg, 210 mg, 700 mg, 2100 mg, or 4200 mg), provided that the first dose amount is lower than the second dose amount. As another example, for a first dose amount that is higher than the second dose amount, the first dose amount can in some embodiments range from 70 mg to 4200 mg (e.g., 70 mg, 210 mg, 700 mg, 2100 mg, or 4200 mg) and the second dose amount can range from 35 mg to 2100 mg (e.g., 35 mg, 70 mg, 210 mg, 700 mg, or 2100 mg), provided that the first dose amount is higher than the second dose amount.

[0098] In some embodiments, when changing from a first dose amount to a second dose amount, the first dose is administered a minimum number of times before the second dose is administered. For example, the first dose can be administered for at least one, at least two, at least three, or at least four 28-day cycles (e.g., where the cycle comprises administering the NKG2D fusion protein once every two weeks or once every four weeks), administered for up to four 28-day cycles (e.g., one, two, three, or four), administered for at least one, at least two, at least three, or at least four 21-day cycles (e.g., where the cycle comprises administering the NKG2D fusion protein once every three weeks), or administered for up to four 21-day cycles (e.g., one, two, three, or four). In some embodiments, the second dose amount is subsequently administered for a minimum number of times. For example, the second dose can be administered for at least one, at least two, at least three, or at least four 28-day cycles (e.g., where the cycle comprises administering the NKG2D fusion protein once every two weeks or once every four weeks), or administered for at least one, at least two, at least three, or at least four 21-day cycles (e.g., where the cycle comprises administering the NKG2D fusion protein once every three weeks). Further exemplary dose adjustments that can be made are described in Section 8.2.4.

[0099] NKG2D fusion proteins can be administered intravenously, for example by intravenous infusion. The length of an infusion can vary, for example based on dose and concentration of the product administered. In some embodiments, a dose of an NKG2D fusion protein is administered over on half hour to three hours, for example over one hour or over two hours. NKG2D fusion proteins can also be administered subcutaneously. To limit or prevent administration-related reactions and/or cytokine release syndrome (CRS), one or more agents can be administered prior to or simultaneous with the NKG2D fusion protein, for example one or more non-steroidal anti-inflammatory drugs, such as acetaminophen, one or more H1/H2 antagonists, such as diphenhydramine (H1 antagonist) and ranitidine (H2 antagonist), one or more corticosteroids, such as dexamethasone or methylprednisolone, or a combination thereof.

[0100] In some embodiments, the NKG2D fusion protein is administered as monotherapy. In other embodiments, the NKG2D fusion protein is administered in combination with one or more additional anti-cancer agents (e.g., one or more chemotherapeutic agents and/or one or more anti-PD-1 agents and/or one or more anti-PD-L1 agents). For example, an NKG2D fusion protein can be administered in combination with a standard of care therapy for a subject’s cancer.

[0101] In some embodiments, the NKG2D fusion protein is administered in combination with or more anti-PD-1 antibodies, for example, Tislelizumab, Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, or Spartalizumab.

[0102] Amino acid sequences of exemplary anti-PD-1 antibodies are set forth in Table A.

[0103] In some embodiments, the NKG2D fusion protein is administered in combination with or more anti-PD-L1 antibodies, for example, Atezolizumab, Avelumab, or Durvalumab.

[0104] Amino acid sequences of exemplary anti-PD-L1 antibodies are set forth in Table B.

[0105] In some embodiments, the NKG2D fusion protein is administered in combination with IL-15 or an IL-15/IL-15Ra heterocomplex (e.g., an IL-15/IL-15Ra heterodimer) (e.g., an IL- 15/IL-Ra heterocomplex disclosed in WO 2007/001677 or WO 2007/084342, the contents of which are incorporated herein by reference in their entireties). Exemplary IL-15/IL-15Ra heterocomplexes (e.g., heterodimers) are additionally described in WO 2021/156720, the contents of which are incorporated herein by reference in their entireties. In some embodiments, an IL-15/IL-15Ra heterodimer comprises IL-15 and IL-15Ra polypeptides having the following amino acid sequences:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDA SIHDTVE NLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:59) (IL- 15 polypeptide)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWT TPSLK CIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQL M PSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQG (SEQ ID NO:60) (IL- 15-Ra polypeptide)

[0106] Additional exemplary IL-15 and IL-15/IL-15Ra amino acid sequences are set forth in Table C.

[0107] In some embodiments, the NKG2D fusion protein is administered in combination with or more anti-TGF-p agents, e.g., anti-TGF-p antibodies, for example NIS793 or fresolimumab.

[0108] Amino acid sequences of exemplary anti-TGF-p antibodies are set forth in Table D.

[0109] The VH, VL, and CDR sequences of NIS793 are described in WO 2012/167143, the contents of which are incorporated herein by reference in their entireties. WO 2012/167143 identifies the heavy chain CDR1 , CDR2, and CDR3 sequences as GGTFSSYA (SEQ ID NO:68), IIPIFGTA (SEQ ID NO:69), and ARGLWEVRALPSVY (SEQ ID NQ:70), respectively, and the light chain CDR1 , CDR2, and CDR3 sequences as DIGSKS (SEQ ID NO:71), EDI, and QVWDRDSDQY (SEQ ID NO:72), respectively. The VH and VL sequence of NIS793 comprise QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANY AQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGLWEVRALPSVYWGQGTLVTVS S (SEQ ID NO:73) and SYELTQPPSVSVAPGQTARITCGANDIGSKSVHWYQQKAGQAPVLWSEDIIRPSGIPERI S GSNSGNTATLTISRVEAGDEADYYCQVWDRDSDQYVFGTGTKVTVLG (SEQ ID NO:74), respectively.

[0110] In some embodiments, the NKG2D fusion protein is administered in combination with an anti-TGF-p antibody having the heavy chain CDR1-CDR3 of SEQ ID NOs:68-70 and light chain CDR1-CDR3 of SEQ ID NO:71 , EDI, and SEQ ID NO:72, respectively. In some embodiments, the NKG2D fusion protein is administered in combination with an anti-TGF-p antibody having a VH and a VL of SEQ ID NOs:73-74, respectively. In some embodiments, the NKG2D fusion protein is administered in combination with an anti-TGF-p antibody having heavy chain and light chain sequences of SEQ ID NOs: 64-65, respectively.

[0111] In some embodiments, a subject having lung cancer, esophageal cancer, or head and neck cancer can be treated with an NKG2D fusion protein in combination with an anti- PD-1 agent, an anti-PD-L1 agent, a platinum-based chemotherapy agent, or a combination thereof. In some embodiments, a subject having renal cancer, e.g., renal cell carcinoma, can be treated with an NKG2D fusion protein in combination with an anti-PD-1 agent, an anti-PD- L1 agent, a VEGFR tyrosine kinase inhibitor, or a combination thereof.

[0112] In some embodiments, a subject having lung cancer (e.g., NSCLC) is treated with an NKG2D fusion protein in combination with Tislelizumab. 8. EXAMPLES

8.1. Example 1 : Determination of human doses of NKG2D fusion protein HH8

8.1.1. Overview

[0113] HH8 is an NKG2D fusion protein comprising two monomers, where each monomer sequence comprises an antibody Fc domain coupled to two NKG2D extracellular receptor domains. The amino acid sequence of the HH8 monomer is set forth as SEQ ID NO:23 (see Table 3). To predict a human dose of HH8, a pharmacokinetic/pharmacodynamic (PK/PD) model was built and parameterized from in vitro and in vivo studies and translated to human. Three mechanisms of action were considered independently:

1) Predictions for intra-tumoral drug concentration were coupled to in vitro measurements of tumor cell killing in a co-culture assay with primary human NK cells to predict in vivo antibody-dependent cellular cytotoxicity (ADCC).

2) Predictions for intra-tumoral drug concentration and receptor occupancy predictions were coupled to in vitro measurements to predict intra-tumoral NK cell NKG2D recovery due to tumor cell membrane neutralization.

3) Predictions for systemic drug concentration combined with predicted MICA (one of the NKG2D ligands) target turnover, determined by allometric scaling from mouse injection studies to human, to predict NK cell NKG2D recovery from systemic soluble ligand neutralization.

8.1.2. Model Overview

[0114] The mathematical model describing the action of HH8 is based on a two- compartment PK model and a receptor-ligand interaction PD model. A schematic of this PK/PD model is shown in FIG. 1.

[0115] In the model, HH8 exists in multiple states, and its concentration is tracked in the central serum compartment (C _c in V _c ), in the peripheral compartment (C _p in V _p ), and in the tumor compartment ( in Vi). HH8 can distribute to the peripheral compartment with macroscopic rate Q12 and the concentration in the tumor compartment is assumed to rapidly distribute at any given time such that the tumor concentration, Ct, is the central serum concentration multiplied by a biodistribution factor (BDF). NKG2D ligand (NKG2D-L) is modeled as synthesized with rate P _syn ,L and eliminated with rate k _e .L from the central serum compartment and described by its concentration in this compartment (L _c ). A HH8 - NKG2D- L complex (C _com p/ _ex ) forms by reversible binding with on-rate k _on and off-rate k _O ft. Early in vitro observations indicated that HH8 binds to monovalent versus multivalent NKG2D-L with a roughly 10x lower affinity (data not shown), highlighting the strong role of avidity in ligand binding. Also, as an intended mechanism of action of HH8 is to perturb soluble NKG2D-L mediated downregulation of NKG2D on NK cells in systemic circulation, it was hypothesized that to achieve reasonable binding affinities to NKG2D on NK cells, NKG2D-L must natively be present in a multi-valent complex. Additionally, some NKG2D-L is secreted via exosomes which implies membrane co-localization (Fernandez-Messina et al., 2012, Front Immunol 3:299). Based on this evidence, it was assumed that HH8 is either unbound or fully bound to two NKG2D-L, and that k _on and kott reflect effective binding rates with avid binding. The rate of clearance of free and NKG2D-L bound HH8 in the central serum compartment is assumed to be the same (k _e ).

[0116] Equations for the model (1-10) are shown below.

Ordinary differential equations of the PK/PD model (Equations 1-4):

Equations 5 - 10 describe derived rate parameters and other modeled quantities:

, CL k _P — — (5)

⁶ Vc

> ln(2) ^l l/2 ~ k (6) Ke,L _r where CL is the clearance term and tm is the calculated half-life of MICA in human. syn k _e LMICAQ (7) off ^on^d (8) where MICAo is the baseline concentration of NKG2D-L and Kd is the dissociation constant describing the binding affinity of HH8 for NKG2D-L. Free HH8 in tumor was modeled as a fraction of that in the central compartment at any time, assuming rapid equilibrium between compartments (Equation 9). The biodistribution factor (BDF) was assumed to be between 10-30% and its variability (or uncertainty) was modeled by sampling from a uniform distribution. Although this value has not been well established for human tumors in a clinical setting, evidence from preclinical studies and physiologically based pharmacokinetic (PBPK) modeling approaches support this range of values (Shah & Betts, 2013, mAbs 5(2):297-305, Deng et al., 2016, mAbs 8(3): 593-603).

C _t = BDF * C _c (9)

To calculate the percentage of NKG2D-L bound to HH8, we use a standard equation describing receptor occupancy (RO), which assumes that the concentration of HH8 in the relevant compartment (C), is in excess compared to ligand.

8.1.3. Modeling steps and process overview

8.1.3.1. Estimating monkey HH8 PK parameters

[0117] HH8 toxicokinetics (TK) were assessed in a 4-week cynomolgus monkey toxicology study. Briefly, HH8 was administered intravenously (i.v.) to male and female cynomolgus monkeys at 30, 100, and 250 mg/kg once perweek (q1w) for four total doses. HH8 pharmacokinetic (PK) parameters (V _c , V _p , CL, Q12) were characterized by fitting a linear two- compartment pharmacokinetic model to the individual animal data using non-linear mixed effects with Monolix 2020R1 (Lixoft). Inter-individual variability (IIV) was modeled on 14 and CL and a proportional error model was used.

8.1.3.2. Estimating mouse MICA terminal elimination rate and half-life

[0118] To estimate the native turnover rate of an NKG2D-L, two studies were performed in which recombinant MICA, one of the family of NKG2D-L’s, was injected into NOD scid gamma (NSG) immunodeficient mice and in Balb/c mice. MICA concentration in serum was then monitored over time and its terminal half-life was calculated. In one study (study 1), 20 pg recombinant human MICA*008 was injected intravenously into Balb/c and NSG mice (n = 2 each). 20 pL blood was collected from the tail vein at 5 min, 30 min, 45 min, 1 h and 4 h post injection. Serum was extracted from blood and assayed for MICA level with Human MICA DuoSet ELISA kit (R&D SYSTEMS DY1300) following the standard manufacturer protocol. MICA clearance was similar in Balb/c and NSG mice. Thus, data from both strains were combined for modeling the half-life of recombinant human MICA in mouse. In a second study (study 2), 10 g recombinant human MICA*008 was injected intravenously into Balb/c mice (n = 6). 20 pL blood was collected from tail vein at 5 min, 15 min, 30 min, 45 min,1 h and 2 h post injection. Serum was extracted from blood and assayed for MICA level with the Human MICA DuoSet ELISA kit (R&D SYSTEMS DY1300) following the standard manufacturer protocol.

[0119] MICA PK was modeled using a standard two-compartmental PK model with linear elimination and parameter fitting to the data and simulations were performed with the IQRTools library (iqrtools.intiquan.com) in R. Although i.v. injection led to two- compartmental, and thus multi-phasic, behavior, it was assumed that early kinetics are related to the exogenous application of MICA and its distribution phase, and the late, terminal kinetics were focused on as the most related to the native elimination rate. The terminal elimination rate, jB, of a two-compartmental PK model in mouse was calculated as: where ki ₂ and k _2i are the transport rates from compartment 1 to 2 and 2 to 1 , respectively, and k _e ,m is the elimination rate from the central compartment. The terminal half-life in mouse, ti/ ₂ ,m, is then calculated as:

8.1.3.3. Translating PK parameters to human

[0120] Translation of parameters estimated in monkeys to humans was performed using single species allometric scaling which has been shown to be appropriate for monoclonal antibodies and leveraged here as HH8 contains an Fc region (Deng et al., 2011 , mAbs 3(1 ) :61 -66; Wang et al., 2016, Biopharm Drug Disp 37:51-65). Scaling exponents used were 1 for V _c and V _p and 0.85 for CL and QI ₂ . Human body weight was assumed to be 70 kg whereas a monkey body weight of 2.79 kg was used as the study average. Given the underlying animal to animal variability, inter-individual variability (I IV) was modeled for and in the monkey and these estimated parameters were propagated forward to the human prediction. Thus, the simulated human PK reflects the uncertainty in parameter estimation from monkey and may not necessarily be representative of typical human PK parameter I IV.

8.1.3.4. PD parameters and translation to human

[0121] In binding assays using engineered 293FT cells overexpressing human NKG2D-Ls, HH8 binds to all expressed NKG2D-L with EC ₅ o values ranging from 0.09 nM to 3.28 nM. The highest of these is MICA with EC ₅ o of 3.28 nM, so by generally representing NKG2D-L with MICA specific parameters, the model is conservative. On-cell binding EC ₅ o values were in good agreement with binding affinity by surface plasmon resonance (SPR) (data not shown), and as such the EC50 value from on-cell binding for MICA was also used as the value of the dissociation constant, Kd. Although kinetic studies were not performed, a typical value for biologies of 10 ⁴ nM' ¹ s ^-1 was assumed. As human MICA was used, it was assumed that this in vitro measurement is reflective of the human in vivo scenario.

[0122] The patient baseline MICA concentration in systemic circulation, MICA ₀ , was estimated in humans to be 0.1 nM. This value is based on baseline MICA levels for a patient treated with autologous tumor vaccination and CTLA-4 blockade (Jinushi et al., 2006, PNAS 103(24):9190-9195), which is believed to be a conservative estimate. However, it was found that model simulations, as they become normalized to baseline MICA levels, are insensitive to this assumed value (simulations not shown).

[0123] Finally, MICA turnover rate, k _e ,L, was translated from mouse injection studies to humans by allometric scaling. In addition to antibody therapeutics, biologies in general have been shown in general to scale allometrically from monkey to humans (Mahmood 2004, J Pharm Sci 93(1 ): 177- 185; Mahmood 2013, Xenobiotica 43(9)774-779; Woo and Jusko, 2007, Drug Metab Dispos 35(9):1672-1678; Kagan et a/., 2010, Pharm Res 27:920-932). Although scaling from mouse is not appropriate for human or humanized monoclonal antibodies because of the poor binding of mouse FcRn to human Fc, FcRn binding does not apply to MICA and therefore it was assumed that MICA turnover observed in mouse studies could be scaled allometrically to human. Assuming standard exponents of 1 for V and 0.85 for CL, the following relationship between elimination rate of MICA in humans, k _e ,i_, and terminal elimination rate, from i.v. injection into mouse was determined:

A human body weight (BW _h ) of 70 kg and a mouse body weight (BW _m ) of 20 g were assumed.

8.1.3.5. Incorporating experimental data to contextualize the model simulations

[0124] To aid interpreting human predictions for the different MoAs, experimental data focused on studying a single MoA in isolation was used to add context. 8.1.3.5.1. ADCC

[0125] As an in vitro measurement of ADCC, a co-culture cell killing assay was used. Briefly, OVCAR-3 human ovarian cancer cells and human primary natural killer (NK) cells from a donor were co-cultured. Cell count was measured overtime by impedance with isotype (negative control) treatment or HH8 treatment at doses ranging from 32 pM to 500 nM.

[0126] To process this kinetic data, a custom script was written in Matlab. For each concentration the time course from HH8 was divided by the impedance measurements from the corresponding isotype group. Next, the region of maximal kill rate, or the largest negative slope of the line, was determined. This maximum negative slope value was then plotted over all tested concentrations and a four-parameter logistic regression dose-response curve was fitted to extract EC ₅ o and EC ₉₀ values. These ECso and EC ₉ o values were then used for comparing intra-tumoral concentrations with those shown in vitro to achieve these kill rate thresholds.

8.1.3.5.2. Soluble ligand neutralization

[0127] As an in vitro measurement of soluble ligand neutralization, an assay culturing human primary NK cells with cancer patient serum was performed. Human peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and resuspended in cR10 assay media. To each well of a 96-well tissue-culture plate, 75,000 PBMCs (22.5 pl) were added with 37.5 pl serum from three melanoma patients (iSpecimen), and 15 pl HH8- DANAPA or isotype control at 6.4 nM, 32 nM and 160 nM. HH8-DANAPA is an Fc-silenced mutant of HH8 that lacks binding to Fc Rs expressed on immune cells, preventing ADCC. The assay was incubated at 37 degrees Celsius + 5% CO2 in a standard tissue culture incubator for 48 hours. Cells were then harvested with 100 pL FACS Buffer and gentle pipetting, washed twice in 200 pL FACS Buffer, and stained with anti-CD4, anti-CD8, anti- CD56 and anti-NKG2D in 100 pL FACS Buffer for 15 min, followed by a wash and fixation. Samples were analyzed on a BD Fortessa. Flow cytometry standard (FCS) data files were analyzed in FlowjoTM (Becton, Dickenson & Company) to estimate the NKG2D median fluorescence intensity (MFI). HH8-DANAPA was able to restore surface NKG2D level on NK cells incubated with melanoma patient serum samples, with the most significant effect seen when using 160 nM.

[0128] Univariate statistical analysis was performed on this data in Prism and identified that there was a statistically significant effect of HH8 on restoring NKG2D surface expression on NK cells versus isotype control with 160 nM and a non-statistically significant, but clear trend, at 32 nM. Assuming HH8 binds to MICA, and other NKG2D-L’s, with Kd = 3.28 nM, the percentage of the ligand in the cancer serum to be neutralized was calculated as 90% at 32 nM and 97% at 160 nM via the following relationship:

100 * c % neutralized — — — — C + K _d

Where C is the drug concentration added in vitro. Although 32 nM was not statistically significant, it was hypothesized that the true threshold concentration for statistically significant effect on restoring NKG2D surface expression on NK cells was between 32 nM and 160 nM, and thus 90-97% NKG2D-L neutralized. 97% neutralized was considered as a firm threshold and the range from 90-97% as a possible threshold range.

8.1.3.5.3. Membrane ligand neutralization

[0129] As an in vitro measurement of membrane NKG2D-L neutralization, data from a coculture assay with primary human NK cells with a MICA*008 overexpressing tumor cell line was leveraged. CT26.WT murine colorectal carcinoma cells were engineered to overexpress human MICA*008. Parental and engineered CT26.WT cells were thawed approximately one week before the assay into cR10 growth media + 5 pg/mL blasticidin. On the day of the assay, cells were washed in cR10 assay media before resuspending in fresh assay media. Human NK cells were isolated, frozen, and thawed on the day of the assay into assay media. On the day of the assay, 50,000 NK cells and 25,000 parental or engineered CT26.WT cells were added to each well of a 96-well flatbottom tissue-culture plate. Dilutions of HH8-DANAPA or isotype control were added at the indicated concentrations, such that the total volume of assay media in each well was 100 pL. The assay was then incubated at 37 degrees Celsius + 5% CO ₂ in a standard tissue culture incubator for 24 hours. Non-adherent cells (consisting mostly of NK cells) were then harvested with 100 pL FACS Buffer and gentle pipetting, washed twice in 200 pL FACS Buffer, and stained with anti-CD45 and anti-NKG2D antibodies in 100 pL FACS Buffer for 15 min, followed by a wash and fixation. Samples were analyzed on a BD Fortessa. FCS files were analyzed in FlowjoTM for the NKG2D MFI of CD45+ cells (pre-gated on forward scatter and side scatter to exclude dead cells, debris, and doublets). Compared to parental cells, CT26.WT cells expressing human MICA*008 induced a reduction in NK cell surface NKG2D when treated with isotype control. Administration of HH8-DANAPA was able to restore NKG2D expression on the surface of NK cells in a dose-dependent manner.

[0130] An EC ₉ o value of 41.5 nM was calculated in Prism assuming a Hill coefficient = 1. Using Equation 10, the percentage of tumor cell membrane NKG2D-L neutralized at the ECg ₀ for NK cell surface NKG2D recovery was calculated to be 92.6%. [0131] Additionally, evidence from an in vivo tumor model expressing human MICA*001 was used to describe NKG2D restoration on intra-tumor NK and CD8+ T-cells after treatment with HH8. Briefly, this was an in vivo study with a syngeneic mouse model where EL4 mouse lymphoblastic cells, either a parental line or cells expressing human MICA*001 , were implanted into mice. Mice were then treated with an isotype control or HH8 dose of 1 , 5, or 20 mg/kg and tumors were harvested to measure NKG2D surface expression on intra- tumoral NK and CD8+ T-cells.

8.1.4. Modeling assumptions and limitations

[0132] The translational modeling approach used to predict the human efficacious dose range and minimal pharmacologically active dose (mPAD) of HH8 was based on the following main assumptions:

• Human PK parameters of HH8 can be predicted from monkey PK parameters via allometric scaling;

• HH8 rapidly distributes between the systemic circulation and intra-tumoral compartment and that its biodistribution factor is similar to that of monoclonal antibodies (which also contain an Fc domain);

• HH8 concentrations are in excess of that of its ligands in relevant compartments;

• HH8, due to its bi-valency, binds to two soluble ligands simultaneously, and this complex is eliminated from the systemic circulation with the same rate as free HH8;

• Human bodyweight of 70 kg, monkey bodyweight of 2.79 kg (average of observed), and mouse bodyweight of 20 g;

• The EC ₅ o value from on-cell binding for MICA is representative of the in vivo dissociation constant, K _d , for all NKG2D-L and a typical k _on value for biologies (10 ⁴ nM’ ¹ S' ¹ ) was assumed;

• MICA turnover observed in mouse studies could be scaled allometrically to human to describe the turnover of the family of NKG2D-L in patients.

[0133] Finally, limitations in the predictive ability of the human HH8 PK/PD model described herein arise from the uncertainty around relationship between the individual mechanisms used for predicting the anticipated human dose and clinical efficacy. 8.1.5. Results

8.1.5.1. Monkey PK model data and fitting

[0134] Results from the 4-week cynomolgus monkey toxicology study indicated that HH8 exhibited linear PK across the dose range, did not display sex differences in PK, and antidrug antibodies (ADA) in two animals did not impact exposure.

[0135] HH8 pharmacokinetic (PK) parameters were characterized by fitting a linear two compartment pharmacokinetic model to the TK data using Monolix 2020R1 . Model fits were reasonable for most of the animals, except one, where an over-prediction of the terminal phase concentration time data was observed. HH8 pharmacokinetic fitted parameter values were reasonable for an Fc-fusion protein (similar to those of monoclonal antibodies with linear clearance; Betts et al., 2018, mAbs 10(5)751-764) and estimated with good confidence (Table 4).

[0136] Based on these estimates, the terminal half-life of HH8 in cynomolgus monkey is approximately 6 days. These parameters were then scaled to human using principles of allometry (Deng et al., 2011 , mAbs 3(1):61-66; Wang et al., 2016, Biopharm Drug Disp 37: 51-65) in order to assess the human pharmacokinetics of HH8.

8.1.5.2. Mouse MICA PK model data and fitting

[0137] To inform the model parameter for NKG2D-L clearance rate, the terminal half-life of MICA was calculated after injection into mice. PK parameters are summarized in Table 5.

[0138] As MICA displayed bi-exponential kinetics in both studies, the terminal half-life of ICA was estimated for each study independently. Due to inter-study variability and uncertainty in parameter estimation, the parameter distributions (defined by estimated value and standard deviation) were re-sampled 1000 times to generate a distribution of terminal half-lives. Consistent with the higher apparent clearance of MICA in study 2, the estimated mouse half-life is lower than that from study 1. Although there is no overlap in the estimated half-life distributions for the two studies, they are on similar timescale, with a total range of approximately 20-65 minutes which is in the range reported for cytokines (Leelahavanichkul et a/., 2011 , Kidney International 80(11):1198-1211).

8.1.5.3. Translating MICA terminal half-life to humans

[0139] To translate the estimated mouse half-life to humans, the allometric scaling procedure outlined in Section 8.1 .3.4 was followed. The human half-life of MICA was estimated to be between 2.5-8.5 hours based on the mouse PK studies. Inter-study variability was larger than inter-individual variability, the highest and lowest observed values between both studies were simulated in the human model as a uniform distribution with lower and upper boundaries set at 3 and 8 hours, respectively.

8.1.5.4. Prediction of human PK/PD

8.1.5.4.1. Human translational PK/PD parameters

[0140] An overview of the parameters used to predict human PK/PD responses are summarized in Table 6. The inter-individual variability I IV reflects the uncertainty in parameter estimation from monkey and may not be representative of typical human PK parameter I IV.

8.1.5.4.2. Human PK simulations

[0141] Using the human parameters shown in Table 6, the PK of free HH8 (HH8 not bound to ligand, MICA, in the serum) was simulated. FIG. 2A shows the simulated free concentration of HH8 in serum for i.v. doses of 70, 210, 700, and 1400 mg Q2W (assuming a bodyweight of 70 kg). The solid line represents the simulations from the parameter point estimates, whereas the range reflects 90% prediction interval based on the I IV on the PK parameters (where here the I IV represents the uncertainty in the estimation of the PK parameters from monkey). Simulations for Q2W, Q3W, and Q4W regimens in units of ng/mL are shown in FIGS. 3AA-3CC. Calculated Cmax and AUC after 1 dose and at steady state are shown in Table 7.

[0142] Although MICA half-life was re-sampled, due to HH8 being far in excess this variability does not contribute to the predicted PK variability. FIG. 2B shows the minimum free concentration over the 2-week dosing interval, Cmin, as a function of dose with colored bars highlighted to represent the doses shown in FIG. 2A. The banded area plotted reflects the 90% prediction interval as plotted in FIG. 2A. Finally, FIG. 2C shows the minimum free concentration over the 2-week dosing interval, Cmin, within the tumor as a function of dose. Compared to FIG. 2B, the 90% prediction interval has been multiplied by the biodistribution factor (BDF) of 10-30% to convert to intra-tumoral concentrations and due to this additional source of uncertainty the prediction bands are wider, as well as concentrations lower than in serum.

[0143] Compared to the NOAEL dose established in cynomolgus monkey of 250 mg/kg, the human PK model predicts exposure margins of 280 and 210 for Cmax and AUC(0-168hr) after a 70 mg dose and 28 and 21 for Cmax and AUC(0-168hr) after a 700 mg dose.

8.1.5.4.3. Human ligand neutralization simulations

[0144] Based on the predicted concentration of free HH8 in the intra-tumoral environment, the neutralization of MICA which is assumed to be membrane bound on tumor cells was predicted using the equation:

100 * c

% neutralized = — — — C + ^K d where Cmin is the minimum concentration of free HH8 in the tumor.

[0145] To reflect the least amount of neutralization present at the end of the 2 week dosing interval, the intra-tumor Cmin values shown in FIG. 2C were used predict the minimum neutralization in the tumor, shown in FIG. 4. Due to the variability in intra-tumoral Cmin, the percent neutralized is similarly variable, most noticeable at lower concentrations where Cmin is similar or less than the Kd value. Therefore, while a 70 mg dose has a wide prediction band of -20-80% neutralized, a 1400 mg dose is predicted to yield a narrower range (-80 to -100%).

[0146] As the model assumes that, in the absence of HH8, MICA is constantly at an equilibrium between synthesis and clearance, the amount of MICA is dynamically changing with time. As such, the percentage of baseline soluble MICA in the systemic circulation that is neutralized with HH8 can be predicted as a function of time. FIGS. 5A-5B show that with all dosing regimens simulated, there is near immediate neutralization of all MICA. However, as HH8 begins to clear out and MICA continues to be produced as a function of being shed or secreted from the intra-tumoral environment into the systemic circulation, the amount of the baseline concentration that is neutralized decreases. As the half-life, and thus k _e ,L, of MICA was modeled as a uniform distribution over a range, FIG. 5A shows the 90% prediction interval from these simulations and, in the absence of additional information as to the most likely parameter values, there is no representative individual simulation plotted on the graph.

[0147] The minimum soluble systemic MICA neutralization over the dosing interval as a function of dose is plotted in FIG. 5B (calculated in the same way that the minimum free serum concentration of HH8 in FIG. 2B was calculated from the values in FIG. 2A). Here, the minimum neutralization, which occurs at the end of the dosing interval, can be considered a conservative representation of efficacy since the predicted neutralization will be higher for earlier time-points of the dosing interval.

8.1.5.4.4. Summary of simulations for key model MOAs [0148] While multiple predictions are shown in FIGS. 2A-5B, FIGS. 6A-6C highlight key model simulations for their relevance to the three major mechanisms of action of HH8 considered for modeling. For the mechanism of intra-tumoral ADCC, the associated model simulation is the intra-tumoral concentration at Cmin. For tumor cell membrane ligand neutralization, the associated model simulation is the intra-tumoral membrane ligand neutralization at Cmin. For systemic soluble ligand neutralization, the associated model simulation is the minimum systemic soluble ligand neutralization. In the next section, experimental evidence was used to assign relevant dose levels for mechanism engagement of each mechanism independently.

8.1.5.4.5. Data to support modeling predictions [0149] As described in Section 8.1.3.5, metrics processed from experimental data were used to add context to the model predictions for the independent MoAs.

[0150] For modeling ADCC, data from the co-culture cell killing assay were used. From fitting a dose response to the maximum killing rate, estimated metrics were EC50 = 3.9 nM (0.44 pg/mL) and EC90 = 70 nM (8. Op g/mL). These two concentrations are overlaid as reference points for the Intra-tumoral ADCC MoA in FIG. 7A and for each of these thresholds, the range of doses that is predicted to reach it is indicated by a double pointed arrow.

[0151] For modeling intra-tumor membrane ligand neutralization, data from the NKG2D downregulation and restoration from primary NK cell and MICA expressing tumor cell coculture were used. At the observed concentration for 90% restoration in NKG2D expression on NK cells (EC90) (41 .5 nM, 4.73 pg/mL), it was predicted that 92.6 percent of cell surface ligand was neutralized. This percentage threshold, labeled as EC90, is overlaid as a reference point for the tumor cell membrane ligand neutralization MoA in FIG. 7B. A double pointed arrow indicates the range of doses predicted to reach this threshold. As additional evidence for this mechanism, in a mouse tumor model, 20 mg/kg HH8 was found to restore NKG2D expression on intratumor NK and CD8+ T-cells, whereas the next lowest does of 5 mg/kg did not. Therefore, the dose range of 5 to 20 mg/kg (350 - 1400 mg for a bodyweight of 70 kg) was additionally indicated as a range for where this mechanism may be engaged.

[0152] For modeling systemic soluble ligand neutralization, data from NK cell culture with cancer patient serum were used. At 160 nM (18.2 pg/mL) HH8, the concentration found to have statistical significance between HH8 and isotype control, 97% of the soluble ligands in the cancer patient serum were predicted to be neutralized and thus a threshold of 97% neutralization is included as a reference line in FIG. 7C, with a double pointed arrow indicating the range of doses that is predicted to reach it. Additionally, at 32 nM (3.6 pg/mL) HH8, there was a clear trend between HH8 and isotype control, although it did not reach statistical significance. At this concentration, 90% of the soluble ligands in the cancer patient serum were predicted to be neutralized. It is, therefore, plausible that the threshold neutralization level lies between 90-97%, and as such this range is overlaid for this MoA in FIG. 7C. A dotted double pointed arrow indicates the range of doses that is predicted to reach 90% neutralization.

8.1.5.4.6. Data to support modeling predictions

[0153] FIGS. 7A-7C shows the model predictions for the three modeled MoAs overlaid with the experimental reference points. For each, shaded regions highlight the dose range(s) in which that mechanism is predicted to be engaged. The dose range varies from mechanism to mechanism (FIG. 7A-7C). The relative contribution of each mechanism to efficacy has not yet been elucidated. The lowest dose for ADCC predicted to achieve intra-tumoral values at EC90 values for in vitro tumor cell killing is 170 mg Q2W. The lowest dose for tumor cell membrane ligand neutralization to achieve neutralization consistent with 90% recovery in NK cell NKG2D expression from co-culture in vitro is 110 mg. The lowest dose for systemic soluble ligand neutralization to achieve soluble ligand neutralization is 1400 mg Q2W based on 97% neutralized, or 700 mg Q2W based on 90% neutralization.

[0154] To explore the impact of longer dosing intervals, FIGS. 8A-8C show a comparison between model simulations and the same key experimental metrics for Q2W versus Q3W dosing. While 90% prediction intervals for intra-tumoral ADCC (FIG. 8A) and tumor cell membrane ligand neutralization (FIG. 8B) are largely overlapping in the upper bound of the prediction interval across those two dosing regimens, the soluble ligand neutralization (FIG. 8C) has predicted ~10% increase with Q2W dosing versus Q3W dosing. Therefore, Q2W dosing is recommended as a starting interval to maximize soluble ligand neutralization and as clinical learnings about the relative importance of each mechanism advance, Q3W and Q4W regimens may be supported.

8.1.5.4.7. Starting dose prediction

[0155] For starting dose prediction, a minimum pharmacologically active dose (mPAD) approach was used to select a dose of 70 mg i.v. Q2W. Briefly, the mPAD approach offers potential benefit for cancer patients at the starting dose, where average concentrations are predicted to achieve EC50-EC85 for ADCC, 60-90% intra-tumoral cell membrane neutralization, and 40-65% systemic soluble ligand neutralization (shown in FIGS. 9A-9C; based on 90% prediction interval of average concentrations, Cavg, during dosing interval, instead of Cmin concentrations).

[0156] Additionally, at a starting dose the time above the stricter thresholds applied for efficacious dose prediction for the different mechanism is predicted to be on the order of days, with the exception of intra-tumoral ADCC predicted to have concentrations surpassing EC50 for multiple weeks (FIGS. 10A-10C). Table 8 summarizes the findings for the 70 mg i.v. Q2W starting dose. Taken together, these simulations highlight the possibility for clinical efficacy at a dose of 70 mg i.v. Q2W based on mechanism engagement.

8.1.5.5. Conclusions

[0157] A multi-compartment PK/PD model was built to describe PK of HH8, as well as the concentration-time profile of its ligands (represented by MICA), their binding reactions in systemic circulation, and in the intra-tumoral environment. To consider the proposed mechanisms of action of HH8, the model simulations focused on three key MoAs: 1) Intratumoral ADCC, 2) Intra-tumoral membrane NKG2D-L neutralization, and 3) Systemic soluble NKG2D-L neutralization. As the relative contribution of these different mechanisms to clinical efficacy is yet to be elucidated, mechanisms were modelled independently and experimental data was leveraged to aid in the interpretation of the results for each.

[0158] Based on this modeling work, HH8 is predicted to achieve meaningful thresholds of intratumoral ADCC at doses greater than 170 mg Q2W, of tumor cell membrane ligand neutralization at doses greater than 110 mg Q2W, and of systemic soluble ligand neutralization at doses between 700-1400 mg Q2W. As the ability of HH8 to modulate the systemic immune system in cancer patients is of interest to evaluate clinically, it is believed that it is appropriate to target an efficacious dose of 700 mg Q2W based on the systemic soluble ligand neutralization predictions. Based on a minimally pharmacologically active dose (mPAD) approach, it is believed that a starting dose of 70 mg Q2W is appropriate to target ADCC EC50-EC85, 60-90% intra-tumoral cell membrane neutralization, and 40-65% systemic soluble ligand neutralization, based on average HH8 concentrations.

8.2. Example 2: A phase l/lb, open-label, multi-center, study of an NKG2D fusion protein in patients with advanced solid tumors

8.2.1. Overview

[0159] A phase l/lb, open-label, multi-center, study of the NKG2D fusion protein HH8 as a single agent is performed to characterize safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary anti-tumor activity of HH8 in adult patients with advanced/metastatic non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), renal cell carcinoma (RCC), and human papilloma virus (HPV)- associated head and neck squamous cell carcinoma (HNSCC). The study consists of a dose escalation part followed by a dose expansion part. For RCC during dose expansion, only the clear cell variant is included.

8.2.2. Study Rationale

[0160] Without being bound by theory, HH8 is believed to have anti-tumor activity by binding to natural killer group 2D ligand (NKG2D-L) (membrane-bound on tumor cells and shed/soluble), via Antibody-dependent Cell-mediated Cytotoxicity (ADCC) and/or reversal of systemic desensitization. The main purpose of this first in human (FIH) study is to characterize safety, tolerability, PK, PD, and preliminary anti-tumor activity of HH8 in adult patients with advanced/metastatic NSCLC, ESCC, RCC, and HPV-associated HNSCC. Dose escalation and expansion includes these four indications. For RCC during dose expansion, only the clear cell variant is included. For NSCLC, ESCC, and HPV-associated HNSCC, PD-L1 expression is used to enrich for immune-inflamed tumors, which contain immune cell infiltration that may enhance response to HH8. Patient populations may be expanded to other biomarker subgroups, including PD-L1 negative patients. HH8 may increase the efficacy of other anti-cancer treatments and this study may be amended to explore HH8 in combination with other anti-cancer treatments.

8.2.3. Objectives and Endpoints

[0161] Objectives and endpoints of the study are summarized in Table 9. 8.2.1. Study Design

[0162] The study consists of a dose escalation part followed by a dose expansion part. Enrollment in both parts is limited to patients with NSCLC, ESCC, RCC, and HPV positive HNSCC. The study design is summarized in FIG. 11 .

8.2.1.1. Dose escalation

[0163] The dose escalation part of the study treats up to approximately 45 patients with NSCLC, ESCC, RCC, or HPV-associated HNSCC. HH8 is initially administered on a Q2W. Alternative dosing schedules include Q3W and Q4W.

[0164] During the dose escalation part of the study, cohorts of 3 to 6 evaluable patients are treated with HH8 single agent until the MTD is reached or a lower RD is established. The MTD and/or RD is identified in one or more of the schedules (Q2W, Q3W, and/or Q4W). An adaptive Bayesian logistic regression model (BLRM) using the escalation with overdose control (EWOC) principle guides dose escalation to determine the MTD and/or RD.

[0165] The study treatment is administered until the patient experiences unacceptable toxicity, progressive disease, discontinues treatment at the discretion of the investigator or the patient, or due to withdrawal of consent.

8.2.1.2. Dose expansion

[0166] The study enters the dose expansion after an MTD(s) and/or RD(s) is determined in the dose escalation. Alternative MTDs/RDs are determined for different dose schedules.

[0167] Once the MTD/RD for a selected dosing schedule is identified for HH8 single agent, approximately 20 patients per indication per dose schedule are treated in each dose expansion group to further evaluate safety and tolerability, and to assess the preliminary anti-tumor activity of HH8.

[0168] The provisional dose expansion groups for the selected dosing schedule are as follows:

• Group 1 : Patients with NSCLC and Programmed death-ligand 1 (PD-L1)>1%

• Group 2: Patients with ESCC and PD-L1>1%

• Group 3: Patients with clear cell RCC

• Group 4: Patients with HPV-associated HNSCC and PD-L1>1%

8.2.2. Study Population

[0169] This study is conducted in patients with advanced or metastatic non-small cell lung cancer, esophageal squamous cell carcinoma, renal cell carcinoma, and HPV-associated head and neck squamous cell carcinoma for whom standard of care therapy for their indication has failed or who are intolerant of or ineligible for approved therapies.

[0170] Patients who meet all the following inclusion and none of the exclusion criteria are included in the study.

8.2.2.1. Inclusion Criteria

[0171] Patients included in this study meet all of the following criteria:

1 . Signed informed consent obtained prior to participation in the study.

2. Patients are willing and able to comply with the protocol for the duration of the study including undergoing treatment, scheduled visits and examinations including follow-up.

3. Adult men and women > 18 years of age.

4. Histologically confirmed and documented advanced malignancies (locally advanced malignancies, non-curable by surgery or radiotherapy and metastatic disease). Disease is measurable, including presence of at least one measurable lesion, as determined by RECIST v1.1.

5. In the opinion of the treating investigator, patients have received, but are not benefitting from standard therapies, be intolerant or ineligible to receive such therapy, or have no standard therapy option. At minimum the following therapies listed below will have been given in the past for the respective disease type, as well as any other therapies deemed to be standard by local/institutional standard.

• Non-small cell lung cancer: Histologically confirmed non-squamous or squamous histology with historic PD-L1 > 1% by local IHC staining. Patients will have received prior treatment that includes anti-PD(L)-1 and a platinum-based chemotherapy regimen, either in combination or in sequence, unless patient is ineligible to receive such therapy. Tumors do not have known activating alterations in EGFR, ALK, ROS1, or RET.

• Esophageal squamous cell carcinoma: Histologically confirmed esophageal squamous cell carcinoma with historic PD-L1 > 1% by local IHC staining. Patients will have received prior treatment that includes a platinum-based chemotherapy regimen and if appropriate, anti- Programmed cell death protein 1 (PD-1) therapy, either in combination or separately, unless patient is ineligible to receive such therapy.

Renal cell carcinoma: Histologically confirmed renal cell carcinoma. For clear cell histology, patients will have received prior treatment including anti-PD-(L)1 and VEGFR TKI, either in combination or in sequence, unless patient was ineligible to receive such therapy. For dose expansion, only patients with clear cell histology are included.

• HPV-associated head and neck squamous cell carcinoma: Histologically confirmed head and neck squamous cell carcinoma with historic positive p16 and PD-L1 > 1% by local IHC staining. Patients will have received prior treatment that includes platinum-based chemotherapy regimen, and if appropriate, anti-PD-1 therapy, either in combination or separately, unless patient was ineligible to receive such therapy.

6. Have a site of disease amenable to biopsy and be a candidate for tumor biopsy. The patient is willing to undergo a new tumor biopsy at screening and during treatment.

7. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 .

8. Patients have a life expectancy of 3 months or more.

8.2.2.2. Exclusion Criteria

[0172] Patients meeting any of the following criteria are not included in this study.

1 . Presence of symptomatic Central Nervous System (CNS) metastases or CNS metastases that require local CNS-directed therapy (such as radiotherapy or surgery), or increasing doses of corticosteroids <2 weeks prior to study entry. Patients with treated symptomatic brain metastases are neurologically stable (for 4 weeks post-treatment and prior to study entry) and at a dose of <10 mg per day prednisone or equivalent for at least 2 weeks before administration of any study treatment.

2. History of allogeneic bone marrow or solid organ transplant.

3. T reatment with any of the following anti-cancer therapies prior to the first dose of study treatment within the stated timeframes:

• < 4 weeks for radiation therapy or limited field radiation for palliation within < 2 weeks prior to the first dose of study treatment.

• < 4 weeks or < 5 half-lives (whichever is shorter) for chemotherapy or biological therapy (including monoclonal antibodies) or continuous or intermittent small molecule therapeutics or any other investigational agent.

< 6 weeks for cytotoxic agents with major delayed toxicities, such as nitrosoureas and mitomycin C. • Patients who have undergone major surgery < 4 weeks prior to first dose of study treatment or who have not recovered for the surgical procedure.

4. Active previously documented or suspected autoimmune disease. Patients with vitiligo, type I diabetes, residual hypothyroidism only requiring hormone replacement, psoriasis not requiring systemic treatment, or conditions not expected to recur are not excluded. Patients previously exposed to anti-PD-1/PD-L1 treatment who are adequately treated for skin rash or with replacement therapy for endocrinopathies are not excluded.

5. Patients with a history of or current interstitial lung disease (ILD) or pneumonitis > Grade 2.

6. Patients who have discontinued prior anti-PD-1 therapy due to an anti-PD-1 -related toxicity.

7. Patients who required discontinuation of treatment due to treatment-related toxicities during prior therapy directed against the same target as the drug under study in this study.

8. Clinically significant cardiac disease or risk factors at screening including any of the following:

• Clinically significant and / or uncontrolled heart disease such as congestive heart failure requiring treatment (NYHA grade > 2), uncontrolled hypertension, defined by blood pressure > 140/90 mmHg at rest (average of 3 consecutive readings) despite medical treatment or clinically significant arrhythmia despite medical treatment.

• QT corrected with Fredericia’s (QTcF) > 470 ms on screening ECG or congenital long QT syndrome

• Acute myocardial infarction or unstable angina pectoris < 3 months prior to study entry.

9. History of severe hypersensitivity reactions to any ingredient of study drug(s) and/or their excipients.

10. Insufficient bone marrow function at screening:

(a) Laboratory value meeting any of the following:

• Absolute Neutrophil Count (ANC) < 1.5 x 10 ⁹ /L

Hemoglobin (Hgb) < 9.0 g/dL (without transfusion support within 7 days prior to the first dose of study drug) Platelets < 75 x 10 ⁹ /L without transfusion support within 7 days prior to the first dose of study drug

OR

(b) Use of hematopoietic colony-stimulating factors (e.g. G-CSF, GM-CSF, M-CSF), thrombopoietin mimetics, or erythroid stimulating agents <2 weeks prior to start of study treatment. If growth factors were initiated more than 2 weeks prior to the first dose of study treatment and the patient is on a stable dose, they can be maintained

11 . Insufficient hepatic or renal function at screening:

• Total bilirubin > 1.5 x ULN, except for patients with Gilbert’s syndrome who are excluded if total bilirubin > 3.0 x ULN or direct bilirubin > 1 .5 x ULN

• Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) > 3 x ULN or > 5 x ULN if liver metastases are present

• Creatinine clearance < 45 mL/min (calculated using Cockcroft-Gault equation)

12. Patients who have not had resolution, except where otherwise stated in the inclusion/exclusion criteria, of all clinically significant toxic effects of prior systemic cancer therapy, surgery, or radiotherapy to Grade <1 (except for alopecia, vitiligo, residual hypothyroidism requiring only hormone replacement or other endocrinopathies adequately treated with replacement therapy, ototoxicity, or peripheral neuropathy (both ototoxicity and peripheral neuropathy) if present, must be < Grade 2)

13. Malignant disease, other than that is being treated in this study. Exceptions to this criterion include the following: malignancies that were treated curatively and have not recurred within two years prior to study treatment; completely resected basal cell and squamous cell skin cancers; any malignancy considered to be indolent and that has never required therapy; and completely resected carcinoma in situ of any type.

14. Patients who are taking a prohibited medication that cannot be discontinued at least seven days prior to the first dose of study treatment and for the duration of the study.

15. Any medical condition that prevents the patient’s participation in the clinical study due to safety concerns or compliance with clinical study procedures.

16. Infections:

• Known history of testing positive for Human Immunodeficiency Virus (HIV) infection.

• Active Hepatitis B (HBV) and / or Hepatitis C (HCV). (a) Active HBV is defined by positive HBsAg and detectable HBV DNA level in serum. Patients with positive HBsAg and HBV DNA level below the limit of quantification can be enrolled with concurrent antiviral therapy.

(b) Active HCV is defined by quantitative HCV RNA results greater than the lower detection limits of the assay.

• Active, documented COVID-19 infection

• Known history of tuberculosis

• Any serious uncontrolled (untreated or unresponsive to treatment) infection (acute or chronic), such as but not limited to those caused by bacteria, viruses, or fungi, confirmed by clinical evidence, imaging, and/or relevant positive laboratory tests (e.g., blood cultures, polymerase chain reaction (PCR) for DNA/RNA, etc).

17. Use of any live or attenuated vaccines against infectious diseases within 4 weeks of initiation of study treatment.

18. Participation in any additional, parallel, investigational drug or device studies.

19. Systemic chronic steroid therapy (>10 mg/day prednisone or equivalent) or any immunosuppressive therapy, other than replacement-dose steroids in the setting of adrenal insufficiency, within 7 days of the first dose of study treatment. Topical, inhaled, and ophthalmic steroids are allowed.

20. Pregnant or breast-feeding (lactating) women, where pregnancy is defined as the state of a female after conception and until the termination of gestation, confirmed by a positive human chorionic gonadotropin (hCG) laboratory test.

21 . Women of child-bearing potential, defined as all women physiologically capable of becoming pregnant, unless they are using highly effective methods of contraception while taking study treatment and for 90 days after stopping study treatment. Women are considered post-menopausal if they have had 12 months of natural (spontaneous) amenorrhea with an appropriate clinical profile (e.g., age appropriate (generally age from 40 to 59 years), history of vasomotor symptoms (i.e., hot flush)). Women are considered not of child bearing potential if they are post-menopausal or have had surgical bilateral oophorectomy (with or without hysterectomy), total hysterectomy or bilateral tubal ligation at least six weeks prior to enrollment on study. In the case of oophorectomy alone, only when the reproductive status of the woman has been confirmed by follow-up hormone level assessment is she considered to be not of child bearing potential. 22. Patients who have previously received therapies targeting NKG2D or NKG2D-L.

8.2.3. Study Treatment and Concomitant Therapies

[0173] For this study, “investigational drug” and "study treatment" refers to HH8. No other treatment beyond investigational drug is included in this trial.

8.2.3.1. Study treatments

[0174] The initial study treatment is defined as intravenous HH8 administered every two weeks. Additional regimens are explored (e.g., Q3W, Q4W).

[0175] In both dose escalation and dose expansion, HH8 is administered by i.v. infusion at the frequency specified over one hour, with flexibility to extend infusion time as clinically indicated. In dose expansion, patients receive HH8 at the regimen specific RD determined in dose escalation.

[0176] Patients do not receive pre-medication to prevent adverse drug/infusion reaction before the first dose of study treatment. If HH8 is associated with infusion-related reactions and/or CRS for a given patient, prophylactic premedication is administered to the patient prior to each of the infusions of HH8. The pre-medication includes acetaminophen, H1/H2 antagonists, and corticosteroids. Premedication is administered for each infusion or discontinued as deemed appropriate for a given patient.

8.2.3.2. Treatment duration

[0177] For the purpose of scheduling and evaluations, a treatment cycle consists of 28 days for patients treated with HH8 (Q2W or Q4W), and 21 days for HH8 Q3W. The treatment period commences on Day 1 of Cycle 1 .

[0178] In the event of disease progression per RECIST v1 .1 , discontinuation of patient is the protocol default. However, clinical experience indicates that objective responses to immunotherapy may follow delayed kinetics and may be preceded by initial apparent radiologic progression, appearance of new lesions, or mixed responses (Wolchok et al., 2009, Clin Cancer Res 15(23)7412-7420). Accordingly, continuation of HH8 treatment beyond disease progression per RECIST v1 .1 for patients who are clinically stable is permitted.

[0179] T o continue study treatment at time of progressive disease per RECIST v1.1 , a patient meets each of the following criteria:

Absence of clinical symptoms or signs indicating clinically significant disease progression. • No decline in performance status.

• Absence of rapid disease progression or threat to vital organs or critical anatomical sites (e.g. CNS metastasis, respiratory failure due to tumor compression, spinal cord compression) requiring urgent alternative medical intervention.

• No significant, unacceptable or irreversible toxicities related to study treatment.

[0180] A patient continues study treatment until the patient experiences unacceptable toxicity, disease progression as per RECIST v1.1 (unless they meet the aforementioned criteria, and consent to receive treatment beyond RECIST v1.1), and/or treatment is discontinued at the discretion of the investigator or the patient, or withdrawal of consent.

8.2.4. Dose Modification

[0181] The starting dose for HH8 in the study is 70 mg administered via i.v. infusion over two hours Q2W. HH8 starting and provisional dose levels that are evaluated during this trial are described in Table 10. Dose escalation continues until the MTD and/or RD is identified.

[0182] Dose escalation is conducted to establish the dose(s) and regimen(s) of single agent HH8 used in the expansion part of the study. Specifically, these are the doses that have the most appropriate benefit-risk as assessed by the review of safety, tolerability, PK, any available efficacy, and PD, taking into consideration the MTD.

[0183] The MTD is the highest dose estimated to have less than 25% risk of causing a DLT during the DLT evaluation period in more than 33% of treated patients. The dose(s) selected for dose expansion is(are) a dose equal to or less than the MTD.

[0184] The dose escalation starts with the dosing schedule of HH8 Q2W. Additional dosing schedule(s) (e.g. Q3W or Q4W) are opened. The starting dose of a new regimen is a dose equal or lower than the highest dose tested in Q2W regimen or the next higher untested dose of Q2W that satisfies the EWOC criteria, whichever is the highest, as declared at the DEM. A separate BLRM is set up to guide the selection of starting dose and dose escalation for any new dosing schedule.

[0185] Intra-patient dose escalation is not performed at any time within the first 4 cycles of treatment. After the 4 ^th cycle is completed, individual patients are considered for treatment at a higher dose of HH8 or on an alternative schedule than the one to which they are initially assigned. In order for a patient to be treated at a higher dose or a transition to an alternative schedule of HH8, he or she must tolerate the assigned dose for at least 2 cycles of therapy (i.e., he or she must not experience any HH8-related toxicity Common Terminology Criteria for Adverse Events (CTCAE) grade > 2 at the lower dose originally assigned). There is no limit to the number of times a patient has his or her dose of HH8 increased.

8.2.5. Results

[0186] Treatment with HH8 shows a positive effect on overall response rate (ORR), disease control rate (DCR), duration of response (DOR), and progression-free survival (PFS).

9. SPECIFIC EMBODIMENTS

[0187] The present disclosure is exemplified by the specific embodiments below.

1 . A method of treating a subject having a cancer comprising administering to the subject a therapeutically effective amount of a dimeric protein comprising two monomers, wherein each monomer comprises: a) a first NKG2D peptide or variant thereof; b) a second NKG2D peptide or variant thereof; c) a first peptide linker connecting said first NKG2D peptide or variant thereof and said second NKG2D peptide or variant thereof; and d) a fragment crystallizable region (Fc region) of an immunoglobulin (Ig).

2. A dimeric protein for use in a method of treating a subject having a cancer, wherein the dimeric protein comprises two monomers, each monomer comprising: a) a first NKG2D peptide or variant thereof; b) a second NKG2D peptide or variant thereof; c) a first peptide linker connecting said first NKG2D peptide or variant thereof and said second NKG2D peptide or variant thereof; and d) a fragment crystallizable region (Fc region) of an immunoglobulin (Ig).

3. The method of embodiment 1 or the dimeric protein for use of embodiment 2, wherein the method comprises administering the dimeric protein at a dose ranging from 20 mg to 5000 mg.

4. The method or dimeric protein for use of any one of embodiments 1 to 3, wherein the method comprises administering the dimeric protein at a dose ranging from 35 mg to 4200 mg.

5. The method or dimeric protein for use of any one of embodiments 1 to 4, wherein the method comprises administering the dimeric protein at a dose ranging from 20 mg to 2500 mg, 35 mg to 70 mg, 35 mg to 210 mg, 35 mg to 700 mg, 35 mg to 2100 mg, 70 mg to 210 mg, 70 mg to 700 mg, 70 mg to 2100 mg, 210 mg to 700 mg, 210 mg to 2100 mg, 700 mg to 2100 mg, or 2100 mg to 4200 mg.

6. The method or dimeric protein for use of any one of embodiments 1 to 5, wherein the method comprises administering the dimeric protein at a dose of 35 mg, 70 mg, 210 mg, 700 mg, 2100 mg, or 4200 mg.

7. The method or dimeric protein for use of any one of embodiments 1 to 6, wherein the method comprises administering the dimeric protein at a dose of 35 mg.

8. The method or dimeric protein for use of any one of embodiments 1 to 6, wherein the method comprises administering the dimeric protein at a dose of 70 mg.

9. The method or dimeric protein for use of any one of embodiments 1 to 6, wherein the method comprises administering the dimeric protein at a dose of 210 mg.

10. The method or dimeric protein for use of any one of embodiments 1 to 6, wherein the method comprises administering the dimeric protein at a dose of 700 mg.

11 . The method or dimeric protein for use of any one of embodiments 1 to 6, wherein the method comprises administering the dimeric protein at a dose of 2100 mg.

12. The method or dimeric protein for use of any one of embodiments 1 to 6, wherein the method comprises administering the dimeric protein at a dose of 4200 mg.

13. The method or dimeric protein for use of any one of embodiments 1 to 12, wherein the method comprises administering the dimeric protein once every two weeks. 14. The method or dimeric protein for use of embodiment 13, wherein the method comprises administering the dimeric protein on day 1 (± 3 days) and day 15 (± 3 days) of a 28-day cycle.

15. The method or dimeric protein for use of any one of embodiments 1 to 12, wherein the method comprises administering the dimeric protein once every four weeks.

16. The method or dimeric protein for use of embodiment 15, wherein the method comprises administering the dimeric protein on day 1 (± 3 days) of a 28-day cycle.

17. The method or dimeric protein for use of any one of embodiments 1 to 16, wherein the method comprises two or more 28-day cycles and/or up to twenty-four 28-day cycles.

18. The method or dimeric protein for use of any one of embodiments 1 to 17, wherein the method comprises three or more 28-day cycles.

19. The method or dimeric protein for use of any one of embodiments 1 to 18, wherein the method comprises four or more 28-day cycles.

20. The method or dimeric protein for use of any one of embodiments 1 to 18, wherein the method comprises six or more 28-day cycles.

21 . The method or dimeric protein for use of any one of embodiments 16 to 20, wherein the method comprises repeating the 28-day cycles until disease progression.

22. The method or dimeric protein for use of any one of embodiments 1 to 12, wherein the method comprises administering the dimeric protein once every three weeks.

23. The method or dimeric protein for use of embodiment 22, wherein the method comprises administering the dimeric protein on day 1 (± 3 days) of a 21 -day cycle.

24. The method or dimeric protein for use of embodiment 22 or embodiment 23, wherein the method comprises two or more 21 -day cycles and/or up to twenty-four 21 -day cycles.

25. The method or dimeric protein for use of any one of embodiments 22 to 24, wherein the method comprises three or more 21 -day cycles. 26. The method or dimeric protein for use of any one of embodiments 22 to 25, wherein the method comprises four or more 21 -day cycles.

27. The method or dimeric protein for use of any one of embodiments 22 to 26, wherein the method comprises six or more 21-day cycles.

28. The method or dimeric protein for use of any one of embodiments 23 to 27, wherein the method comprises repeating the 21-day cycles until disease progression.

29. The method or dimeric protein for use of any one of embodiments 1 to 28, wherein the method comprises administering the same dose of the dimeric protein at each administration.

30. The method or dimeric protein for use of any one of embodiments 1 to 28, wherein the method comprises administering the dimeric protein one or more times at a first dose amount and subsequently administering the dimeric protein one or more times at a second dose amount.

31 . The method or dimeric protein for use of embodiment 30, wherein the first dose amount is lower than the second dose amount.

32. The method or dimeric protein for use of embodiment 31 , wherein the first dose amount and second dose amount are within the range 20 mg to 5000 mg.

33. The method or dimeric protein for use of embodiment 31 , wherein the first dose amount and second dose amount are within the range 35 mg to 4200 mg.

34. The method or dimeric protein for use of embodiment 31 , wherein the first dose amount and second dose amount are within the range 20 mg to 2500 mg.

35. The method or dimeric protein for use of embodiment 31 , wherein the first dose amount and second dose amount are within the range 35 mg to 2100 mg.

36. The method or dimeric protein for use of any one of embodiments 31 to 34, wherein the first dose amount ranges from 35 mg to 2100 mg.

37. The method or dimeric protein for use of embodiment 36, wherein the first dose amount ranges from 35 mg to 700 mg. 38. The method or dimeric protein for use of any one of embodiments 31 to 36, wherein the first dose amount is 35 mg, 70 mg, 210 mg, 700 mg, or 2100 mg.

39. The method or dimeric protein for use of embodiment 38, wherein the first dose amount is 35 mg.

40. The method or dimeric protein for use of embodiment 38, wherein the first dose amount is 70 mg.

41 . The method or dimeric protein for use of embodiment 38, wherein the first dose amount is 210 mg.

42. The method or dimeric protein for use of embodiment 38, wherein the first dose amount is 700 mg.

43. The method or dimeric protein for use of embodiment 38, wherein the first dose amount is 2100 mg.

44. The method or dimeric protein for use of any one of embodiments 31 to 43, wherein the second dose amount ranges from 70 mg to 4200 mg.

45. The method or dimeric protein for use of embodiment 44, wherein the second dose amount ranges from 70 mg to 2100 mg.

46. The method or dimeric protein for use of any one of embodiments 31 to 44, wherein the second dose amount is 70 mg, 210 mg, 700 mg, 2100 mg, or 4200 mg.

47. The method of dimeric protein for use of embodiment 46, wherein the second dose amount is 70 mg

48. The method of dimeric protein for use of embodiment 46, wherein the second dose amount is 210 mg.

49. The method of dimeric protein for use of embodiment 46, wherein the second dose amount is 700 mg.

50. The method of dimeric protein for use of embodiment 46, wherein the second dose amount is 2100 mg.

51 . The method of dimeric protein for use of embodiment 46, wherein the second dose amount is 4200 mg. 52. The method or dimeric protein for use of embodiment 30, wherein the first dose amount is higher than the second dose amount.

53. The method or dimeric protein for use of embodiment 52, wherein the first dose amount and second dose amount are within the range 20 mg to 5000 mg.

54. The method or dimeric protein for use of embodiment 52, wherein the first dose amount and second dose amount are within the range 35 mg to 4200 mg.

55. The method or dimeric protein for use of embodiment 52, wherein the first dose amount and second dose amount are within the range 20 mg to 2500 mg.

56. The method or dimeric protein for use of embodiment 52, wherein the first dose amount and second dose amount are within the range 35 mg to 2100 mg.

57. The method or dimeric protein for use of any one of embodiments 52 to 54, wherein the first dose ranges from 70 mg to 4200 mg.

58. The method or dimeric protein for use of any one of embodiments 52 to 56, wherein the first dose ranges from 70 mg to 2100 mg.

59. The method or dimeric protein for use of any one of embodiments 52 to 58, wherein the first dose amount is 70 mg, 210 mg, 700 mg, 2100 mg, or 4200 mg.

60. The method of dimeric protein for use of embodiment 59, wherein the first dose amount is 4200 mg.

61 . The method of dimeric protein for use of embodiment 59, wherein the first dose amount is 2100 mg.

62. The method of dimeric protein for use of embodiment 59, wherein the first dose amount is 700 mg.

63. The method of dimeric protein for use of embodiment 59, wherein the first dose amount is 210 mg.

64. The method of dimeric protein for use of embodiment 59, wherein the first dose amount is 70 mg.

65. The method or dimeric protein for use of any one of embodiments 52 to 64, wherein the second dose amount ranges from 35 mg to 2100 mg. 66. The method or dimeric protein for use of embodiment 65, wherein the second dose amount ranges from 35 mg to 700 mg.

67. The method or dimeric protein for use of any one of embodiments 52 to 65, wherein the second dose amount is 35 mg, 70 mg, 210 mg, 700 mg, or 2100 mg.

68. The method or dimeric protein for use of embodiment 67, wherein the second dose amount is 35 mg.

69. The method or dimeric protein for use of embodiment 67, wherein the second dose amount is 70 mg.

70. The method or dimeric protein for use of embodiment 67, wherein the second dose amount is 210 mg

71 . The method or dimeric protein for use of embodiment 67, wherein the second dose amount is 700 mg.

72. The method or dimeric protein for use of embodiment 67, wherein the second dose amount is 2100 mg.

73. The method or dimeric protein for use of any one of embodiments 30 to 72, wherein the first dose amount is administered for at least one 28-day cycle or at least one 21 -day cycle.

74. The method or dimeric protein for use of any one of embodiments 30 to 72, wherein the first dose amount is administered for at least two 28-day cycles or at least two 21 -day cycles.

75. The method or dimeric protein for use of any one of embodiments 30 to 72, wherein the first dose amount is administered for at least three 28-day cycles or at least three 21 -day cycles.

76. The method or dimeric protein for use of any one of embodiments 30 to 75, wherein the first dose amount is administered for at least four 28-day cycles or at least four 21 -day cycles.

77. The method or dimeric protein for use of any one of embodiments 30 to 76, wherein the second dose amount is administered for at least one 28-day cycle or at least one 21 -day cycles. 78. The method or dimeric protein for use of any one of embodiments 30 to 76, wherein the second dose amount is administered for at least two 28-day cycles or at least two 21 -day cycles.

79. The method or dimeric protein for use of any one of embodiments 1 to 78, wherein the cancer is an NKG2D ligand expressing cancer.

80. The method or dimeric protein for use of embodiment 79, wherein the cancer comprises a solid tumor or a liquid tumor.

81 . The method or dimeric protein for use of any one of embodiments 1 to 80, wherein the cancer comprises a solid tumor.

82. The method or dimeric protein for use of any one of embodiments 1 to 81 , wherein the cancer comprises a solid tumor with an immune-inflamed phenotype.

83. The method or dimeric protein for use of any one of embodiments 1 to 82, wherein the subject has PD-L1 expressing tumor cells.

84. The method or dimeric protein for use of any one of embodiments 1 to 83, wherein the subject has PD-L1 expression in > 1% of tumor cells.

85. The method or dimeric protein for use of any one of embodiments 1 to 83, wherein the subject has PD-L1 expression in < 1% of tumor cells.

86. The method or dimeric protein for use of any one of embodiments 1 to 85, wherein the cancer is advanced.

87. The method or dimeric protein for use of any one of embodiments 1 to 86, wherein the cancer is locally advanced.

88. The method or dimeric protein for use of any one of embodiments 1 to 87, wherein the cancer is Stage III cancer.

89. The method or dimeric protein for use of any one of embodiments 1 to 86, wherein the cancer is metastatic.

90. The method or dimeric protein for use of any one of embodiments 1 to 86 and 89, wherein the cancer is Stage IV cancer. 91 . The method or dimeric protein for use of any one of embodiments 1 to 90, wherein the cancer is not curable by surgery.

92. The method or dimeric protein for use of any one of embodiments 1 to 91 , wherein the cancer is not curable by radiotherapy.

93. The method or dimeric protein for use of any one of embodiments 1 to 92, wherein the subject has at least one measurable cancer lesion as determined by RECIST 1.1 criteria prior to administration of the dimeric protein.

94. The method or dimeric protein for use of any one of embodiments 1 to 93, wherein the subject has received one or more standard of care therapies for the cancer.

95. The method or dimeric protein for use of any one of embodiments 1 to 94, wherein the subject is receiving one or more standard of care therapies but is not receiving a clinical benefit from the one or more standard of care therapies and/or the subject has received one or more standard of care therapies but has not received a clinical benefit from the one or more standard of care therapies.

96. The method or dimeric protein for use of any one of embodiments 1 to 95, wherein the subject is intolerant of or ineligible for a standard of care therapy.

97. The method or dimeric protein for use of any one of embodiments 1 to 96, wherein the cancer is a lung cancer, an esophageal cancer, a renal cancer, a head and neck cancer, a pancreatic cancer, a gastric cancer, a colorectal cancer, an ovarian cancer, an endometrial cancer, a biliary tract cancer, a liver cancer, a breast cancer, a prostate cancer, a stomach cancer, a bladder cancer, a hepatocellular carcinoma (HCC), a nasopharyngeal carcinoma (NPC), a melanoma, a plasma cell cancer, a bone cancer, a soft tissue cancer, a glioblastoma multiforme, an astrocytoma, or a hematological cancer.

98. The method or dimeric protein for use of any one of embodiments 1 to 97, wherein the cancer is a lung cancer, an esophageal cancer, a renal cancer, or a head and neck cancer.

99. The method or dimeric protein for use of any one of embodiments 1 to 98, wherein the cancer is a lung cancer.

100. The method or dimeric protein for use of embodiment 99, wherein the lung cancer is non-squamous lung cancer. 101. The method or dimeric protein for use of embodiment 99, wherein the lung cancer is squamous lung cancer.

102. The method or dimeric protein for use of any one of embodiments 99 to 101 , wherein the lung cancer is non-small cell lung cancer.

103. The method or dimeric protein for use of any one of embodiments 99 to 102, wherein the subject has received treatment with (i) an anti-PD-1 and/or anti-PD-L1 agent and (ii) a platinum-based chemotherapy agent, in combination or in sequence.

104. The method or dimeric protein for use of any one of embodiments 99 to 103, wherein the cancer does not have an activating alteration in EGFR, ALK, ROS1, or RET.

105. The method or dimeric protein for use of any one of embodiments 1 to 98, wherein the cancer is an esophageal cancer.

106. The method or dimeric protein for use of embodiment 105, wherein the esophageal cancer is esophageal squamous cell carcinoma.

107. The method or dimeric protein for use of embodiment 105 or embodiment 106, wherein the subject has received treatment with (i) an anti-PD-1 and/or anti-PD-L1 agent and (ii) a platinum-based chemotherapy agent, in combination or in sequence.

108. The method or dimeric protein for use of any one of embodiments 1 to 98, wherein the cancer is a renal cancer.

109. The method or dimeric protein for use of embodiment 108, wherein the kidney cancer is renal cell carcinoma.

110. The method or dimeric protein for use of embodiment 108 or embodiment 109, wherein the kidney cancer is clear cell renal cell carcinoma.

111. The method or dimeric protein for use of any one of embodiments 108 to 110, wherein the subject has received treatment with (i) an anti-PD-1 and/or anti-PD-L1 agent and (ii) a VEGFR tyrosine kinase inhibitor, in combination or in sequence.

112. The method or dimeric protein for use of any one of embodiments 1 to 98, wherein the cancer is a head and neck cancer. 113. The method or dimeric protein for use of embodiment 112, wherein the head and neck cancer is head and neck squamous cell carcinoma.

114. The method or dimeric protein for use of embodiment 112 or embodiment 113, wherein the head and neck cancer is human papilloma virus-associated head and neck cancer.

115. The method or dimeric protein for use of any one of embodiments 112 to 114, wherein the subject has p16 positive tumor cells.

116. The method or dimeric protein for use of any one of embodiments 112 to 115, wherein the subject has received treatment with (i) an anti-PD-1 and/or anti-PD-L1 agent and (ii) a platinum-based chemotherapy agent, in combination or in sequence.

117. The method or dimeric protein for use of any one of embodiments 1 to 97, wherein the cancer is hematological cancer.

118. The method or dimeric protein for use of embodiment 117, wherein the cancer is a Hodgkin’s lymphoma, a non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), or multiple myeloma.

119. The method or dimeric protein for use of any one of embodiments 1 to 118, wherein the method comprises administering the dimeric protein as monotherapy.

120. The method or dimeric protein for use of any one of embodiments 1 to 118, wherein the method comprises administering the dimeric protein in combination with one or more anti-cancer agents.

121. The method or dimeric protein for use of embodiment 120, wherein the one or more anti-cancer agents comprise an anti-PD-1 agent.

122. The method or dimeric protein for use of embodiment 121 , wherein the anti- PD-1 agent is an anti-PD-1 antibody.

123. The method or dimeric protein for use of embodiment 122, wherein the anti- PD-1 antibody is Tislelizumab, Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, or Spartalizumab. 124. The method or dimeric protein for use of embodiment 122 or embodiment 123, wherein the anti-PD-1 antibody is Tislelizumab.

125. The method or dimeric protein for use of embodiment 122 or embodiment 123, wherein the anti-PD-1 antibody is Nivolumab.

126. The method or dimeric protein for use of embodiment 122 or embodiment 123, wherein the anti-PD-1 antibody is Pembrolizumab.

127. The method or dimeric protein for use of embodiment 122 or embodiment 123, wherein the anti-PD-1 antibody is Cemiplimab.

128. The method or dimeric protein for use of embodiment 122 or embodiment 123, wherein the anti-PD-1 antibody is Dostarlimab.

129. The method or dimeric protein for use of embodiment 122 or embodiment 123, wherein the anti-PD-1 antibody is Spartalizumab.

130. The method or dimeric protein for use of any one of embodiments 120 to 129, wherein the one or more anti-cancer agents comprise an anti-PD-L1 agent.

131. The method or dimeric protein for use of embodiment 130, wherein the anti- PD-L1 agent is an anti-PD-L1 antibody.

132. The method or dimeric protein for use of embodiment 130, wherein the anti- PD-L1 antibody is Atezolizumab, Avelumab, or Durvalumab.

133. The method or dimeric protein for use of embodiment 131 or embodiment 132, wherein the anti-PD-L1 antibody is Atezolizumab.

134. The method or dimeric protein for use of embodiment 131 or embodiment 132, wherein the anti-PD-L1 antibody is Avelumab.

135. The method or dimeric protein for use of embodiment 131 or embodiment 132, wherein the anti-PD-L1 antibody is Durvalumab.

136. The method or dimeric protein for use of any one of embodiments 120 to 135, wherein the one or more anti-cancer agents comprise IL-15. 137. The method or dimeric protein for use of any one of embodiments 120 to 136, wherein the one or more anti-cancer agents comprise an IL-15/IL- 15Ra heterocomplex (e.g., having an amino acid sequence of the IL-15 and IL-15/IL-15Ra heterocomplex in Table C).

138. The method or dimeric protein for use of any one of embodiments 120 to 137, wherein the one or more anti-cancer agents comprise an IL-15/IL- 15Ra heterodimer comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:59 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:60.

139. The method or dimeric protein for use of any one of embodiments 120 to 138, wherein the one or more anti-cancer agents comprise an anti-TGB-p agent.

140. The method or dimeric protein for use of embodiment 139, wherein the anti- TGB-p agent is an anti-TGB-p antibody.

141. The method or dimeric protein for use of embodiment 140, wherein the anti- TGB-p antibody is NIS793 or Fresolimumab.

142. The method or dimeric protein for use of embodiment 140 or embodiment 141 , wherein the anti-TGB-p antibody is NIS793.

143. The method or dimeric protein for use of embodiment 140 or embodiment 141 , wherein the anti-TGB-p antibody is Fresolimumab.

144. The method or dimeric protein for use of any one of embodiments 1 to 143, wherein the method comprises administering the dimeric protein by intravenous infusion.

145. The method or dimeric protein for use of any one of embodiments 1 to 144, wherein the method comprises administering the dimeric protein by intravenous infusion over one half hour to three hours.

146. The method or dimeric protein for use of any one of embodiments 1 to 145, wherein the method comprises administering the dimeric protein by intravenous infusion over one hour.

147. The method or dimeric protein for use of any one of embodiments 1 to 145, wherein the method comprises administering the dimeric protein by intravenous infusion over two hours. 148. The method or dimeric protein for use of any one of embodiments 1 to 143, wherein the method comprises administering the dimeric protein by subcutaneous injection.

149. The method or dimeric protein for use of any one of embodiments 1 to 148, wherein the method further comprises prophylactic administration of one or more agents for administration-related reactions and/or CRS prior to administration of the dimeric protein.

150. The method or dimeric protein for use of embodiment 149, wherein the one or more agents for administration-related reactions and/or CRS comprise one or more nonsteroidal anti-inflammatory drugs (optionally acetaminophen), one or more H1/H2 antagonists, one or more corticosteroids, or a combination thereof.

151. The method or dimeric protein for use of any one of embodiments 1 to 150, wherein said first NKG2D peptide or variant thereof and said second NKG2D peptide or variant thereof have identical amino acid sequences.

152. The method or dimeric protein for use of any one of embodiments 1 to 150, wherein said first NKG2D peptide or variant thereof and said second NKG2D peptide or variant thereof have different amino acid sequences.

153. The method or dimeric protein for use of any one of embodiments 1 to 152, wherein said first NKG2D peptide or variant thereof comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 1 , 2, 3 or 4.

154. The method or dimeric protein for use of any one of embodiments 1 to 153, wherein said first NKG2D peptide or variant thereof comprises an amino acid sequence of SEQ ID NO: 1 , 2, 3 or 4.

155. The method or dimeric protein for use of any one of embodiments 1 to 154, wherein said second NKG2D peptide or variant thereof comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 1 , 2, 3 or 4.

156. The method or dimeric protein for use of any one of embodiments 1 to 155, wherein said second NKG2D peptide or variant thereof comprises an amino acid sequence of SEQ ID NO: 1 , 2, 3 or 4.

157. The method or dimeric protein for use of any one of embodiments 1 to 156, wherein said first peptide linker is a glycine-serine peptide linker. 158. The method or dimeric protein for use of any one of embodiments 1 to 157, wherein said first peptide linker is represented by the formula (GGGS) _n (SEQ ID NO: 10), wherein n is 1 , 2, 3, 4, or 5.

159. The method or dimeric protein for use of any one of embodiments 1 to 158, wherein said first peptide linker is (GGGS) ₂ (SEQ ID NO: 11).

160. The method or dimeric protein for use of any one of embodiments 1 to 159, wherein said Fc region comprises an Fc region of human immunoglobulin G (IgG).

161. The method or dimeric protein for use of any one of embodiments 1 to 160, wherein said Fc region comprises an Fc region of human lgG1.

162. The method or dimeric protein for use of any one of embodiments 1 to 161 , wherein said Fc region comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 12, 13, 14, 15, or 16.

163. The method or dimeric protein for use of any one of embodiments 1 to 162, wherein said Fc region comprises the amino acid sequence of SEQ ID NO: 12, 13, 14, 15, or 16.

164. The method or dimeric protein for use of any one of embodiments 1 to 163, wherein said monomer comprises, from N-terminus to C-terminus, said first NKG2D peptide or variant thereof, said first peptide linker, said second NKG2D peptide or variant thereof and said Fc region.

165. The method or dimeric protein for use of embodiment 164, wherein said second NKG2D peptide or variant thereof is directly fused with said Fc region without a peptide linker.

166. The method or dimeric protein for use of embodiment 164, wherein said second NKG2D peptide or variant thereof is linked with said Fc region via a second peptide linker.

167. The method or dimeric protein for use of any one of embodiments 1 to 163, wherein said monomer comprises, from N-terminus to C-terminus, said Fc region, said first NKG2D peptide or variant thereof, said first peptide linker, and said second NKG2D peptide or variant thereof. 168. The method or dimeric protein for use of embodiment 167, wherein said Fc region is directly fused with said first NKG2D peptide or variant thereof without a peptide linker.

169. The method or dimeric protein for use of embodiment 167, wherein said Fc region is linked with said first NKG2D peptide or variant thereof via a second peptide linker.

170. The method or dimeric protein for use of any one of embodiments 1 to 169, wherein said monomer further comprises one or two additional NKG2D peptides or variants thereof.

171. The method or dimeric protein for use of any one of embodiments 1 to 170, wherein said monomer comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, and 40.

172. The method or dimeric protein for use of any one of embodiments 1 to 170, wherein said monomer comprises an amino acid sequence of any one of SEQ ID NOs: 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, and 40.

173. The method or dimeric protein for use of any one of embodiments 1 to 170, wherein each monomer comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:23.

174. The method or dimeric protein for use of any one of embodiments 1 to 170, wherein each monomer comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23.

175. The method or dimeric protein for use of any one of embodiments 1 to 170, wherein each monomer comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:23.

176. The method or dimeric protein for use of any one of embodiments 1 to 170, wherein each monomer comprises an amino acid sequence having 100% sequence identity to SEQ ID NO:23.

177. The method or dimeric protein for use of any one of embodiments 1 to 176, wherein said two monomers are covalently linked. 178. The method or dimeric protein for use of any one of embodiments 1 to 177, wherein said two monomers are linked through a Cys-Cys bridge.

179. The method or dimeric protein for use of any one of embodiments 1 to 178, wherein the method comprises periodically administering the dimeric protein until disease progression, optionally as measured by RECIST 1.1 criteria.

180. The method or dimeric protein for use of any one of embodiments 1 to 179, wherein the method comprises periodically administering the dimeric protein until clinically significant disease progression.

181. The method or dimeric protein for use of any one of embodiments 1 to 180, wherein the method comprises periodically administering the dimeric protein until a decline in ECOG performance status.

182. The method or dimeric protein for use of any one of embodiments 1 to 181 , wherein the method prolongs progression-free survival.

183. The method or dimeric protein for use of any one of embodiments 1 to 182, wherein the subject is at least 18 years of age.

184. The method or dimeric protein for use of any one of embodiments 1 to 183, wherein the subject has an ECOG performance status score of <1 .

185. The method or dimeric protein for use of any one of embodiments 1 to 184, wherein the subject has not previously received a therapy targeting NKG2D.

186. The method or dimeric protein for use of any one of embodiments 1 to 185, wherein the subject has not previously received a therapy targeting NKG2D-L.

187. A combination comprising a dimeric protein and one or more anticancer agents for use in a method of treating a subject having a cancer as described in any of one of embodiments 120 to 143 or any one of embodiments 144 to 186 when depending from any of one of embodiments 120 to 143.

10. CITATION OF REFERENCES

[0188] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.