Human therapy with il-18 immunocytokines

Abstract

The present disclosure relates to methods of treating cancer in humans with immunocytokine compositions comprising antibodies or antigen binding fragments specific for PD-1 and IL-18 polypeptides. Also described herein are pharmaceutical compositions of such immunocytokine compositions.

Description

WSGR Docket No. 56146-744.601HUMAN THERAPY WITH IL-18 IMMUNOCYTOKINESCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 699,651 filed September 26, 2024, and U.S. Provisional Application No. 63 / 706,873 filed October 14, 2024, each of which applications are incorporated herein by reference in their entirety.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 September 3, 2025, is named 56146_744_601_SL.xml and is 188,450 bytes in size.BRIEF SUMMARY

[0003] Described herein are methods of treating cancer in a human subject in need thereof with immunocytokine compositions which contain an anti-PD-1 antibody or antigen binding fragment thereof (e.g., LZM-009 (a.k.a. Lipustobart)) linked to an IL-18 polypeptide (e.g., SEQ ID NO: 30). Also described herein are pharmaceutical compositions of said immunocytokine compositions.

[0004] In some embodiments, the instant disclosure relates to the identification of a therapeutic dose level of an immunocytokine composition (e.g., Composition A described herein) effective to treat cancer in human subjects. In some embodiments, the dose level is in the range of about 1 microgram / kg to about 1000 microgram / kg. In some embodiments, the dose level is about 30 microgram / kg, about 100 microgram / kg, about 300 microgram / kg, or about 450 microgram / kg In some embodiments, the dose level is in the range of about 1 microgram / kg to about 1000 microgram / kg. In some embodiments, the dose level is about 30 microgram / kg, about 100 microgram / kg, about 200 microgram / kg, about 300 microgram / kg, or about 450 microgram / kg In some embodiments, the dose level is about 100 microgram / kg. In some embodiments, the dose level is about200 microgram / kg. In some embodiments, the dose level is about 300 microgram / kg In some embodiments, a therapeutic dose of an immunocytokine composition described herein (e.g., Composition A) is a dose which is substantially lower (e.g., at least 2-fold lower, or more) than a dose which causes a substantial risk of serious adverse side effects. In some embodiments, the instant disclosure describes optimal PK parameters (e.g., Cmax, AUC, etc.) to be achieved in order to impart therapeutic effects of the immunocytokines described herein with minimal risk of side effects.WSGR Docket No. 56146-744.601

[0005] In some embodiments, the instant disclosure also relates to the identification of specific indications of cancer or populations of patients in which the immunocytokine compositions of the instant disclosure (e.g., Composition A described herein) are particularly effective. In some embodiments, the instant disclosure provides that the immunocytokine compositions described herein (e.g., Composition A) are particularly useful in cancers which are resistant to or refractory to treatment with other anti-cancer agents. In some embodiments, the immunocytokine compositions described herein (e.g., Composition A) are useful in treating patients who are refractory to or otherwise unresponsive to checkpoint inhibitor therapies (e.g., therapies which comprise an immune checkpoint inhibitor alone or in combination with other agents, wherein the immune checkpoint inhibitor is not comprised into an immunocytokine composition with an IL- 18 polypeptide as described herein). In some embodiments, the immunocytokine compositions described herein (e.g., Composition A) are effective in treating cancer in patients who have failed to respond to a previous treatment with an immune checkpoint inhibitor (e.g. an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA4 antibody, etc.). In some embodiments, the immunocytokine compositions described herein are particularly useful in the treatment of “hot’ or “warm” tumor types (i.e., those having a high degree of immune cell infiltration in the tumor microenvironment, such as tumor-infiltrating lymphocytes). In some embodiments, the immunocytokine compositions described herein can also be useful in the treatment of “cold” tumor types which exhibit lower levels of immune cell infiltration in the tumor microenvironment, such as by acting a strong activator of the immune cells which are present in the tumor microenvironment.

[0006] In another aspect, the instant disclosure also relates to pharmaceutical compositions comprising the immunocytokine compositions described herein (e.g., Composition A). In some embodiments, the pharmaceutical compositions described herein provide substantial stability to the immunocytokine compositions described herein, thus allowing for the transport and storage of the immunocytokine compositions to the desired location for administration to subjects.

[0007] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.WSGR Docket No. 56146-744.601INCORPORATION BY REFERENCE

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGURE 1A illustrates a non-limiting mechanism of action of an anti-PD-1 antibody / IL- 18 immunocytokine provided herein, wherein an activated T cell shows enhanced activation through concurrent blockade of PD-1 and stimulation by IL-18.

[0010] FIGURE IB illustrates a non-limiting mechanism of action of an anti-PD-Ll antibody / IL- 18 immunocytokine provided herein, wherein the immunocytokine disrupts PD -LI / PD-1 interaction and effectuates IL-18R signaling.

[0011] FIGURE 1C illustrates a non -limiting mechanism of action of an anti-PD-Ll antibody / IL-18 immunocytokine acting on an immune cell expressing both IL-18R and PD-L1.

[0012] FIGURE ID illustrates an immunocytokine composition provided herein with two different protein payloads (e.g., IL-18 and IL-2) linked to an antibody (e.g., PD-L1 antibody) acting on an immune cell with simultaneous activation of two different receptors (e.g., IL-18R and IL-2R).

[0013] FIGURE 2 shows a representative immunocytokine composition with a drug antibody ratio of 1 (DARI).

[0014] FIGURE S shows an illustration of a site selective introduction of a conjugation handle to an antibody as provided herein.

[0015] FIGURE 4 illustrates the mechanism of action of IL- 18 on IFNy and IL- 18BP production, and IL-18 inhibitory activity by IL-18BP.

[0016] FIGURE S shows a schematic of a synthetic route which can be used to synthesize an IL- 18 polypeptide for conjugation to an antibody.

[0017] FIGURE 6 shows a schematic representation of coupling of a bifunctional probe (also referred to as bifunctional linking reagent herein) to an IL-18 polypeptide provided herein.

[0018] FIGURE ? shows exemplary reaction scheme which can be used to prepare antibody / IL- 18 immunocytokine compositions provided herein with drug antibody ratio of 1 (top) or 2 (bottom).WSGR Docket No. 56146-744.601

[0019] FIGURE 8 shows a reverse phase HPLC chromatogram of a purified IL-18 / anti-PD-1 antibody (LZM009) as provided herein with a DAR of 1 attached at residue K248 of the Fc region of the antibody (EU numbering).

[0020] FIGURE 9 shows a size exclusion HPLC chromatogram of a purified IL-18 / anti-PD-1 antibody (LZM009) as provided herein with a DAR of 1 attached at residue K248 of the Fc region of the antibody (EU numbering).

[0021] FIGURE 10 shows a mass spectrometry trace (Q-TOF) of a purified IL- 18 / anti-PD-1 antibody (LZM009) as provided herein with a DAR of 1 attached at residue K248 of the Fc region of the antibody (EU numbering).

[0022] FIGURE 11 shows the RP-HPLC chromatograms of purified composition A (detection: 280 nm).

[0023] FIGURE 12 shows the Analytical SEC -HPLC chromatograms of purified composition A (detection: 280 nm).

[0024] FIGURE 13 shows the Q-TOF mass spectrometry deconvolution chromatogram of purified composition A.

[0025] FIGURE 14A - 14B show plots measuring ability of the unmodified and of conjugated anti-PDl antibodies to bind with human PD1 / CD279 ligand, with the figure showing ELISA signal on the y-axis and dosage of the biotinylated PD-1 protein on the x-axis.

[0026] FIGURE 14A Shows the unconjugated reference antibodies. Tested in this figure are Pembrolizumab, LZM-009, Nivolumab, Atezolizumab, Durvalumab, and Avelumab.

[0027] FIGURE 14B Shows the conjugated antibodies. Tested in this figure are compositions A and composition B.

[0028] FIGURE 15A - 15B show plots measuring ability of the unmodified and of conjugated antibodies to bind with human PD-L1 / B7-H1 ligand, with the figure showing ELISA signal on the y-axis and dosage of the biotinylated PD-L1 protein on the x-axis.

[0029] FIGURE 15A shows the unconjugated reference antibodies. Tested in this figure are Pembrolizumab, LZM-009, Nivolumab, Atezolizumab, Durvalumab, and Avelumab.

[0030] FIGURE 15B shows the conjugated antibodies. Tested in this figure are compositions A and composition B.

[0031] FIGURE 16 shows plots measuring ability of the unmodified and of conjugated antibodies to bind to human PD-L1 / B7-H1 ligand, with the figure showing netBioLayer interferometry shift in nanometer on the y-axis and time of incubation dosage of the biotinylated PD-L1 protein on the x-axis. The figure shows mean ELISA signal on the y -axis and dosage of the human Fc gammaWSGR Docket No. 56146-744.601 receptors on the x-axis. The unconjugated reference antibodies are Pembrolizumab and LZM-009. The conjugated antibodies tested is Compositions A.

[0032] FIGURE 17A - 17B show plots measuring ability of the unmodified and of conjugated anti-PDl antibodies to interfere with PD1 / PDL1 pathway, with the figure showing normalized luminescence intensity of effector cells NFAT-Lucia reporter on the y-axis and dosage of the unmodified and of conjugated anti-PDl antibodies on the x-axis.

[0033] FIGURE 17A shows the unconjugated reference antibodies are Pembrolizumab, LZM- 009, Nivolumab, Atezolizumab, Durvalumab, and Avelumab.

[0034] FIGURE 17B shows the conjugated antibodies tested .Tested in this figure are compositions A and composition B.

[0035] FIGURE 18A shows plots measuring ability of the unmodified and of conjugated antibodies to bind to human Fc gamma receptor I (CD64). The figure shows mean ELISA signal on the y-axis and dosage of the human Fc gamma receptors on the x-axis. The unconjugated reference antibodies are LZM-009 and Atezolizumab. The conjugated antibodies tested are Compositions A and B.

[0036] FIGURE 18B shows plots measuring ability of the unmodified and of conjugated antibodies to bind to human Fc gamma receptor Illa (CD 16). The figure shows mean ELISA signal on the y-axis and dosage of the human Fc gamma receptors on the x-axis. The unconjugated reference antibodies are LZM-009 and Atezolizumab. The conjugated antibodies tested are Compositions A and B.

[0037] FIGURE 19 shows plots measuring ability of the unmodified and of conjugated antibodies to bind to human Fc neonatal receptor. The figure shows mean AlphaLISA® signal on the y-axis and dosage of the human Fc neonatal receptor (FcRn) on the x-axis. The unconjugated reference antibodies are LZM-009 and Atezolizumab. The conjugated antibodies tested are Compositions A and B.

[0038] FIGURE 20 shows plots measuring the levels PD-1 and PD-L1 surface expression on NK92 cells.

[0039] FIGURE 21 shows plots measuring ability of the unconjugated IL-18 variants and corresponding IL- 18 immunocytokines to stimulate the secretion of IFNy by NK92 cells. The figure shows mean IFNy AlphaLISA® signal on the y-axis and dosage of the unconjugated IL-18 variants and corresponding IL-18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL-18 wild-type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. Corresponding IL-18 immunocytokines tested are Compositions A, B, C, and D.WSGR Docket No. 56146-744.601

[0040] FIGURE 22 shows plots measuring the levels PD-1 and PD-L1 surface expression on KG-1 cells.

[0041] FIGURE 23 shows plots measuring the ability of the unconjugated IL-18 variants and corresponding IL-18 immunocytokines to stimulate the secretion of IFNyby parental PD-1ne§ative(round symbols, dotted lines) and by engineered PD-lP°sitiveKG-l cells (squared symbols, solid lines). The figure shows mean I Ny LEGENDplex™ signal on the y-axis and dosage of the unconjugated IL-18 variants and corresponding IL- 18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL- 18 wild-type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. Corresponding IL-18 immunocytokines tested are Compositions A, B, C, andD.

[0042] FIGURE 24 shows plots measuring the ability of the unconjugated IL-18 variants and corresponding IL-18 immunocytokines to bind to the human IL-18 Binding Protein (IL-18BP). The figure shows mean free IL-18BP AlphaLISA® signal on the y-axis and dosage of the unconjugated IL-18 variants and corresponding IL- 18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL- 18 wild-type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. Corresponding IL-18 immunocytokines tested are Compositions A, B, C, and D.

[0043] FIGURE 25 shows plots measuring the ability of the human IL-18 Binding Protein to inhibit the secretion of I Nyby NK92 cells stimulated with 2nM of unconjugated IL-18 variants and corresponding IL- 18 immunocytokines. The figure shows mean IFNy AlphaLISA® signal on the y-axis and dosage of the human IL-18 Binding Protein on the x-axis. The unconjugated IL-18 variants are native IL-18 wild-type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. Corresponding IL-18 immunocytokines tested are Compositions A, B, C, and D.

[0044] FIGURE 26A shows a plot describing the effect of unmodified PD-1 antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the growth ofMC38 syngeneic colon carcinoma tumors in hPDl C57BL / 6 mice. The figure shows mean tumor volume on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A tested as a single agent at 0.3 and 1 mg / kg as two weekly i.v. injections. (n=9 ; mean ± SEM).

[0045] FIGURE 26B shows a plot describing the effect of unmodified PD -LI antibodies and of IL-18 polypeptide conjugated PD-L1 antibody on the growth of MC38 syngeneiccolon carcinoma tumors in hPDl C57BL / 6 mice. The figure shows mean tumor volume on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition B tested as a single agent at 1 and 3 mg / kg as two weekly i.v. injections. (n=9 ; mean ± SEM).

[0046] FIGURE 27A shows a plot describing the effect of unmodified PD-1 antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the body weight of MC38 syngeneic colon carcinoma tumor-bearing hPDl C57BL / 6 mice. The figure shows mean body weight change onWSGR Docket No. 56146-744.601 the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A tested as a single agent at 0.3 and 1 mg / kg as two weekly i.v. injections. (n=9 ; mean ± SEM).

[0047] FIGURE 27B shows a plot describing the effect of unmodified PD -LI antibodies and of IL-18 polypeptide conjugated PD-L1 antibody on the body weight of MC38 syngeneic colon carcinoma tumor-bearing hPDl C57BL / 6 mice. The figure shows mean body weight change on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition B tested as a single agent at 1 and 3 mg / kg as two weekly i.v. injections. (n=9 ; mean ± SEM).

[0048] FIGURE 28A shows a plot describing the effect of unmodified PD-1 antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the growth ofMC38 syngeneic colon carcinoma tumors in hPDl C57BL / 6 mice. The figure shows mean tumor volume on the y-axis and time on the x-axis. The immunocytokine tested in this figure are composition A tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as two weekly i.v. injections. As a control, Her2 -targeted immunocytokine composition C was applied at 0.5 mg / kg as a single agent and in combination with LZM-009 anti-PD-1 antibody at 1 mg / kg (n=9 ; mean ± SEM).

[0049] FIGURE 28B shows a plot describing the effect of unmodified PD-1 antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the growth ofMC38 syngeneic colon carcinoma tumors in hPDl C57BL / 6 mice. The figure shows the mean tumor volume on day 17 post treatment initiation on the y-axis. The immunocytokine tested in this figure are composition A tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as two weekly i.v. injections. As a control, Her2 -targeted immunocytokine composition C was applied at 3 mg / kg as a single agent and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9; One-way Anova test *** P- vahie<0.001, ** P-value<0.01, * P-value<0. 1, ns not significant, TGI: Tumor Growth Inhibition).

[0050] FIGURE 28C shows a plot describing the effect of unmodified PD-1 antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the growth ofMC38 syngeneic colon carcinoma tumors in hPDl C57BL / 6 mice. The figure shows the tumor volume of each individual animal on the y-axis and time on the x-axis. The immunocytokine tested in this figure are composition A tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as two weekly i.v. injections. As a control, Her2 -targeted immunocytokine composition C was applied at 0.5 mg / kg as a single agent and in combination with LZM-009 anti-PD-1 antibody at 1 mg / kg (n=9; CR: Complete Response).

[0051] FIGURE 29 shows a plot describingthe effect of unmodified PD -LI antibodies and of IL- 18 polypeptide conjugated PD-1 antibody on the on the body weight of MC38 syngeneic colon carcinoma tumor-bearing hPDl C57BL / 6 mice. The figure shows mean body weight change on the y-axis and time on the x-axis. The immunocytokine tested in this figure are Composition A tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as two weekly i.v. injections. As a control,WSGR Docket No. 56146-744.601Her2 -targeted immunocytokine composition C was applied at 0.5 mg / kg as a single agent and in combination with LZM-009 anti-PD-1 antibody at 1 mg / kg (n=9 ; mean ± SEM).

[0052] FIGURE 30 shows a plot describing the effect of unmodified PD -LI antibodies and of IL- 18 polypeptide conjugated PD- 1 antibody on the survival of MC38 syngeneic colon carcinoma tumor-bearing hPDl C57BL / 6 mice The immunocytokine tested in this figure are Composition A tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as two weekly i.v. injections. As a control, Her2 -targeted immunocytokine composition C was applied at 0.5 mg / kg as a single agent and in combination with LZM-009 anti-PD-1 antibody at 1 mg / kg (n=9 ; mean ± SEM ; CR: Complete response).

[0053] FIGURE 31 A shows a plot describing the effect of unmodified PD-1 antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the growth of B16F10 syngeneic melanoma tumors in hPDl C57BL / 6 mice. The figure shows mean tumor volume on the y -axis and time on the x-axis. The immunocytokine tested in this figure are composition A tested as a single agent at 0.3, 1, and 3 mg / kg as two weekly i.v. injections. As a control, Her2 -targeted immunocytokine composition C was applied at 3 mg / kg as a single agent and in combination with LZM-009 anti- PD-1 antibody at 10 mg / kg (n=9 ; mean ± SEM).

[0054] FIGURE 31B shows a plot describing the effect of unmodified PD-1 antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the growth of B16F10 syngeneic melanoma tumors in hPDl C57BL / 6 mice. The figure shows the mean tumor volume on day 10 post treatment initiation on the y-axis. The immunocytokine tested in this figure are composition A tested as a single agent at 0.3, 1, and 3 mg / kg as two weekly i.v. injections. As a control, Her2- targeted immunocytokine composition C was applied at 3 mg / kg as a single agent and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9; TGI: Tumor Growth Inhibition).

[0055] FIGURE 31C shows a plot describing the effect of unmodified PD-1 antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the growth of B16F10 syngeneic melanoma tumors in hPDl C57BL / 6 mice. The figure shows the tumor volume of each individual animal on the y-axis and time on the x-axis. The immunocytokine tested in this figure are composition A tested as a single agent at 0.3, 1, and 3 mg / kg as two weekly i.v. injections. As a control, Her2- targeted immunocytokine composition C was applied at 3 mg / kg as a single agent and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9; CR: Complete Response).

[0056] FIGURE 32 shows a plot describing the effect of unmodified PD -LI antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the body weight of Bl 6F 10 syngeneic melanoma tumor-bearing hPDl C57BL / 6 mice. The figure shows mean body weight change on the y -axisWSGR Docket No. 56146-744.601 and time on the x-axis. The immunocytokine tested in this figure are Composition A tested as a single agent at 0.3, 1, and 3 mg / kg as two weekly i.v. injections. As a control, Her2 -targeted immunocytokine composition C was applied at 3 mg / kg as a single agent and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9 ; mean ± SEM).

[0057] FIGURE 33 shows a plot describing the effect of unmodified PD -LI antibodies and of IL-18 polypeptide conjugated PD-1 antibody on the survival of B16F10 syngeneic melanoma tumor-bearing hPDl C57BL / 6 mice. The immunocytokine tested in this figure are Composition A tested as a single agent at 0.3, 1, and 3 mg / kg as two weekly i.v. injections. As a control, Hei2- targeted immunocytokine composition C was applied at 3 mg / kg as a single agent and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9 ; mean ± SEM ; CR: Complete response).

[0058] FIGURE 34 shows plots measuring the levels PD-1 surface expression on wild type NK- 92 cells and on NK-92 cells transduced with human PD-1.

[0059] FIGURE 35 (left panel) shows plots measuring the ability of the unconjugated IL-18 variants and corresponding IL- 18 immunocytokines to stimulate the secretion oflFNyby parental PD-lne§ative(round symbols, dotted lines) and by engineered PD-lP°sitiveNK-92 cells (squared symbols, solid lines). The figure shows mean IFNy AlphaLISA® signal on the y-axis and dosage of the unconjugated IL- 18 variants and corresponding IL- 18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL-18 wild-type (SEQ ID NO:1) and SEQ ID NO: 30. Corresponding IL-18 immunocytokines tested are Compositions A and C.

[0060] FIGURE 35 (right panel) shows plots measuring the ability of the human IL-18 Binding Protein to inhibit the secretion of IFNy by NK92 cells stimulated with 2nM of unconjugated IL- 18 variants and corresponding IL- 18 immunocytokines by parental PD-1ne§ative(round symbols, dotted lines) and by engineered PD-lP°sitiveNK-92 cells (squared symbols, solid lines). The figure shows mean IFNy AlphaLISA® signal on the y-axis and dosage of the human IL-18 Binding Protein on thex-axis. The unconjugated IL-18 variants are native IL-18 wild-type (SEQ ID NO: 1) and SEQ ID NO: 30. Corresponding IL- 18 immunocytokines tested are Compositions A and C.

[0061] FIGURE 36 shows plots measuring the ability of the unconjugated IL-18 variants and corresponding IL-18 immunocytokines to stimulate the secretion of lFNyby parental PD-1ne§atlve(round symbols, dotted lines) and by engineered PD-lP°sitiveNK-92 cells (squared symbols, solid lines) in the absence (black plain symbols) or presence (grey open symbols) of an excess of anti- PD-1 antibody. The figure shows mean IFNy LEGENDplex™ signal on the y-axis and dosage of the unconjugated IL-18 variants and corresponding IL- 18 immunocytokines on the x-axis. TheWSGR Docket No. 56146-744.601 unconjugated IL-18 variants are native IL-18 wild-type (SEQ ID NO:1) and SEQ ID NO: 30. Corresponding IL-18 immunocytokines tested are Compositions A and C.

[0062] FIGURE 37A shows a plot describing plasma exposure of Total (bound and unbound to IL-18BP) and free (unbound to IL-18BP) composition A in hPD-1 transgenic C57BL / 6 mice implanted with MC38 syngeneic colon carcinoma tumors. The figure shows plasma concentrations on the y-axis, and time on the x-axis. The immunocytokine tested is Compositions A tested as a single agent at 0.25 mg / kg as a single i.v. injection. (n=3 per time point ; mean ± SEM).

[0063] FIGURE 37B shows a plot describing tumor exposure of Total (bound and unbound to IL-18BP) and free (unbound to IL-18BP) composition A in hPD-1 transgenic C57BL / 6 mice implanted with MC38 syngeneic colon carcinoma tumors. The figure shows tumor concentrations on the y-axis, and time on the x-axis. The immunocytokine tested is Compositions A tested as a single agent at 0.25 mg / kg as a single i.v. injection. (n=3 per time point ; mean ± SEM).

[0064] FIGURE 38 shows a plot describing the levels of surface expression of PD-1 and IL-18 receptor alpha in the blood and tumors of MC38 tumor bearing hPDl humanized C57BL / 6 mice treated with vehicle. The figure shows the median fluorescence intensity of PD-1 (upper panel) and of IL-18 receptor alpha (bottom panel) surface staining the y-axis, and the subset of immune cell on the x-axis. (48h and 7 days after i.v. Injection of formulation buffer, n=5 per time point).

[0065] FIGURE 39 shows a plot describingthe effect of PD-1 targeted IL-18 immunocytokine on the expansion of CD4+ T-cells, CD8+ T-cells, macrophages, andNatural Killer (NK) cells in the blood and tumors of MC38 tumor bearing hPDl humanized C57BL / 6 mice after treatment. The figure shows the frequency of indicated immune cell subsets within CD45+leukocytes on the y-axis, and time on the x-axis. The immunocytokine tested is Compositions A tested as a single agent at 0.25 mg / kg as a single i.v. injection. (n=3 per time point ; mean ± SEM).

[0066] FIGURE 40 shows a plot describingthe effect of PD-1 targeted IL- 18 immunocytokine on the expansion of naive (CD62Lhi§hCD44low), central memory (CD62Lhi§hCD44hl«h) effector memory (CD62Lne§atlveCD44hi§h) CD8+ T-cells in the blood and tumors of MC38 tumor bearing hPDl humanized C57BL / 6 mice after treatment. The figure shows the frequency of indicated immune cell subsets within CD45+ leukocytes on the y-axis, and time on the x-axis. The immunocytokine tested is Compositions A tested as a single agent at 0.25 mg / kg as a single i.v. injection. (n=3 per time point ; mean ± SEM).

[0067] FIGURE 41 shows a plot describingthe effect of PD-1 targeted IL- 18 immunocytokine on the expression of CD69 surface marker on CD4+ T-cells, CD8+ T-cells, and Natural Killer (NK) cells in the blood and tumors of MC38 tumor bearing hPDl humanized C57BL / 6 mice afterWSGR Docket No. 56146-744.601 treatment. The figure shows the median fluorescence intensity of CD69 surface staining the y - axis, and time on the x-axis. The immunocytokine tested is Compositions A tested as a single agent at 0.25 mg / kg as a single i.v. injection. (n=3 per time point ; mean ± SEM).

[0068] FIGURE 42 shows a plot describingthe effect of PD-1 targeted IL-18 immunocytokine on the expression of IL-18 receptor alpha on the surface of CD4+ T-cells, CD8+ T-cells, and Natural Killer (NK) cells in the blood and tumors of MC38 tumor bearing hPDl humanized C57BL / 6 mice after treatment. The figure shows the median fluorescence intensity of IL-18 receptor alpha surface staining the y -axis, and time on the x-axis. The immunocytokine tested is Compositions A tested as a single agent at 0.25 mg / kg as a single i.v. injection. (n=3 per time point ; mean ± SEM).

[0069] FIGURE 43 shows a plot describingthe effect of PD-1 targeted IL-18 immunocytokine on levels of cytokines in blood and tumors of in hPD-1 transgenic C57BL / 6 mice implanted with MC3 8 syngeneic colon carcinoma tumors. The figure shows plasma and tumor concentrations of IFNy, GM-CSF, TNFa, and IL-6 on the y-axis, and time on the x-axis. The IL- 18 immunocytokine tested is Compositions A tested as a single agent at 0.25 mg / kg as a single i.v. injection. (n=3 per time point ; mean ± SEM).

[0070] FIGURE 44 shows a plot describing the effect of depleting antibody treatment on leukocytes populations of blood andtumors of in hPD-1 transgenic C57BL / 6 mice implanted with MC38 syngeneic colon carcinoma tumors. The figure shows the frequency of indicated immune cell subsets within CD45+ leukocytes on the y-axis, and depleting antibody treatment on the x- axis. The IL- 18 immunocytokine tested is Compositions A tested as a single agent at 0.25 mg / kg as a single i.v. injection. (n=3 per time point ; mean ± SEM).

[0071] FIGURE 45 shows a plot describing the effect CD4+ T-cells, CD8+ T-cells, Macrophages, and Natural Killer (NK) cells depletion on the anti-tumoral activity of PD-1 targeted IL-18 immunocytokine in MC38 tumor bearing hPDl humanized C57BL / 6 mice after treatment. The figure shows the volume of MC38 tumors on the y-axis, and time on the x-axis. The immunocytokine tested is Compositions A tested as a single agent at 0.5 mg / kg as a single i.v. injection. (n=7 per group ; mean ± SEM).

[0072] FIGURE 46 shows a plot describing the effect CD4+ T-cells, CD8+ T-cells, Macrophages, and Natural Killer (NK) cells depletion on the anti-tumoral activity of PD-1 targeted IL-18 immunocytokine in MC38 tumor bearing hPDl humanized C57BL / 6 mice 12 days after treatment. The figure shows the volume of MC38 tumors on the y-axis, and treatment received by mice on the x-axis. The immunocytokine tested is Compositions A tested as a singleWSGR Docket No. 56146-744.601 agent at 0.5 mg / kg as a single i.v. injection. (n=7 per group ; mean ± SEM ; TGI: Tumor Growth Inhibition).

[0073] FIGURE 47 shows a plot describing the effect CD4+ T-cells, CD8+ T-cells, Macrophages, and Natural Killer (NK) cells depletion on the anti-tumoral activity of PD-1 targeted IL-18 immunocytokine in MC38 tumor bearing hPDl humanized C57BL / 6 mice after treatment. The figure shows the volume of MC38 tumors on the y-axis, and time on the x-axis. The immunocytokine tested is Compositions A tested as a single agent at 0.5 mg / kg as a single i.v. injection. (n=7 per group; CR: Complete Response).

[0074] FIGURE 48 shows a plot describingthe effect CD4+ T-cells, CD8+ T-cells, macrophages, and Natural Killer (NK) cells depletion on the anti-tumoral activity of PD-1 targeted IL-18 immunocytokine in MC38 tumor bearing hPDl humanized C57BL / 6 mice after treatment. The figure shows the percentage of surviving mice within each treatment group the y -axis, and time on the x-axis. The immunocytokine tested is Composition A tested as a single agent at 0.5 mg / kg as a single i.v. injection. (n=7 per group).

[0075] FIGURE 49 shows plasma levels of Composition A at various time points in a non -human primate (NHP) study followingthe indicated single dose of Composition A. Results are shown in ng / mL.

[0076] FIGURE 50 shows plasma levels of both “free” Composition A (notbound to IL-18BP) and Composition A complexed with IL-18BP in an NHP study. The results show virtually no Composition A was complexed with IL-18BP.

[0077] FIGURE 51 shows quantification of anti-PD-1 antibody in plasma samples of NHPs measured by capturing material using PD-1 or anti-IL-18. The results from both capture methods are virtually identical, indicating all or nearly all of Composition A remained intact (e.g., no cleavage of the linker between the antibody and the IL-18 polypeptide).

[0078] FIGURE 52 shows levels of IL-18R / PD-1 double positive T cells following administration of Composition A at various dose levels in NHPs.

[0079] FIGURE 53 shows an overview of a human study design for assessing the effect of Composition A on human subjects.

[0080] FIGURE 54 shows an overview of the schedule of Composition A administration according to a study design described herein.

[0081] FIGURE 55 shows an overview of dose escalation of Composition A according to a study design described herein.

[0082] FIGURE 56 shows a schedule of assessments of human subjects receiving Composition A according to the study design described herein.WSGR Docket No. 56146-744.601

[0083] FIGURE 57 shows a schedule showing time points at which samples will be taken from subjects of the study for assessment of pharmacokinetic, blood biomarkers, and / or anti-drug antibodies.DETAILED DESCRIPTION

[0084] Disclosed herein are methods of treating cancer in human subjects with antibodies or antigen binding fragments specific for PD-1 linked to IL-18 polypeptides in immunocytokine compositions. In some embodiments, the use of the immunocytokine compositions described herein is effective in the treatment of a variety of cancers, in particular those which are unresponsive to current standard-of-care therapies. Thus, in certain embodiments, the instant methods are directed to methods of treating cancer in subjects where the cancer is unresponsive or refractory to other therapies, such as immune checkpoint inhibitor therapies alone or in combination with other agents, wherein the immune checkpoint inhibitor is not comprised into an immunocytokine composition with an IL-18 polypeptide as described herein.

[0085] In some instances, the immunocytokine compositions provided herein are useful as potent stimulators of one or more immune cell types (e.g., T cells, macrophages, etc.). In some embodiments, the immunocytokine compositions can actby one or more modes of action. In some embodiments, the immunocytokine compositions (e.g., anti -PD-1 / IL-18 immunocytokines) are effective in the treatment of cancer in the subject.

[0086] In some embodiments, the antibody of the immunocytokine composition allows for targeting of the immunocytokine composition to an immune cell (e.g., a PD-1+immune cell, such as a CD8+T cell). In some embodiments, the immunocytokine composition can inhibit an activity of the immune cell associated antigen (e.g., inhibiting a checkpoint interaction such as a PD-1 / PD- L1 interaction) through binding to the immune cell associated antigen. In some embodiments, the immunocytokine compositions induce IFNyproductionin immune cells (e.g., T cells orNK cells). The antibody or antigen binding fragment-IL-18 immunocytokine compositions of the disclosure can have synergistic efficacy and improved tolerability by a subject. In some embodiments, the antibody or antigen binding fragment-IL-18 immunocytokine compositions can significantly reduce the therapeutic dose of the antibody or antigen binding fragment, the IL-18 polypeptide, or both for a subject with a disease, such as a cancer, as compared to a treatment with one or both entities individually or in combination. In some embodiments, the immunocytokine compositions provided herein are associated with fewer side effects than administration of one or both entities individually or in combination, potentially due to the targeting nature of the antibodies for an immune cell.WSGR Docket No. 56146-744.601

[0087] An exemplary, non-limiting mechanism of action of an immunocytokine provided herein is shown in FIGURE 1A. In the exemplary embodiment, the immunocytokine composition comprises an anti-PD-1 antibody as the antibody or antigen binding fragment of the immunocytokine composition. In this embodiment, the anti-PD-1 antibody portion of the immunocytokine selectively binds to PD-1 present on the surface of an activated T cell (e.g., a CD8+T cell). This binding prevents the checkpoint interaction of PD-1 and PD-L1 / 2, thus preventing attenuation of activity of the T cell. Concomitantly, the IL-18 portion of the immunocytokine, which is effectively in a high local concentration near the T-cell due to the linkage, further activates the T cell through IL-18R signaling. While the exemplary embodiment shows the mechanism of action of an anti-PD-1 antibody, antibodies or antigen binding fragments specific for other immune antigens provided herein can function according to a similar mechanism.

[0088] Another exemplary, non-limiting mechanism of action of an anti-PD-Ll antibody / IL-18 immunocytokine is shown in FIGURE IB. In the exemplary embodiment, the anti-PD-Ll antibody portion of the immunocytokine binds to PD-L1 expressed on the surface of a tumor cell. When a T cell comes into contact with the tumor cell, an interaction between PD-1 on the T cell andPD-Ll on the cell is blocked, preventing attenuation ofthe activity of the T cell. Additionally, the IL-18 portion of the immunocytokine is free to signal through IL-18R on the immune cell, thereby inducing production of IFNy and further activation of the immune cell. Though this exemplary embodiment is demonstrated for PD-L1, other immune antigens provided herein in immunocytokines may display similar mechanisms of action. FIGURE 1C shows a comparable mechanism of action of an anti-PD-Ll / IL-18 immunocytokine acting on a T cell which expresses IL-18R and PD-L1. FIGURE ID shows another mechanism of action of an additional embodiment described herein wherein the antibody of the immunocytokine contains two different protein payloads (e.g., two different cytokines) which act on different receptors of the immune cell.

[0089] Also disclosed herein are methods of manufacturing the immunocytokine compositions provided herein. In some embodiments, the immunocytokine compositions are prepared using chemical linkers which can attach the two moieties of immunocytokine composition to each other at pre-selected sites of each moiety with high fidelity. In some embodiments, the methods provided herein can be used on a wide variety of antibodies or antigen binding fragments in order to rapidly and easily generate a wide variety of immune antigen specific antibody or antigen binding fragments linked to IL- 18 polypeptides as immunocytokines. In some embodiments, the methods can be used on readily commercially available antibodies to allow for rapid linking withWSGR Docket No. 56146-744.601IL-18 polypeptides provided herein. One non-limiting illustration of an immunocytokine as provided herein is shown in FIGURE 2, which depicts an IL-18 polypeptide linked to an antibody as provided herein with a point of attachment of the linker to the IL-18 polypeptide at a side chain of a residue and to a side chain of a residue in the Fc region.

[0090] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications ofthis present disclosure, which are encompassed within its scope.

[0091] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.

[0092] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Anti-PD-1 Antibodies Linked to IL-18 Polypeptides as Immunocytokines

[0093] Provided herein are antibodies and antigen binding fragments which binds to PD-1 linked to IL-18 polypeptides as immunocytokine compositions. The immunocytokine compositions provided herein are effectivefor simultaneously deliveringthe IL-18 polypeptide andthe antibody or antigen binding fragment to a target cell, such as an immune cell, preferably in tumor microenvironment of the cell. This simultaneous delivery of both agents to the same cell has numerous benefits, including improved IL-18 polypeptide selectivity, enhanced therapeutic potential of the IL-18 polypeptide, and minimized risk of side effects from administering IL-18 therapies. In some embodiments, the immunocytokine compositions act through multiple modes of action, includingwithoutlimitation disruptingan activity of the immune cell associated antigen (e.g., immune checkpoint evasion) and / or enhanced activation of immune cells in or around a tumor microenvironment. Thus, it is predicted that human therapies with anti-PD-1 / IL-18 immunocytokine compositions as described herein will provide many advantages over other treatment options, including in subjects who have failed immune checkpoint inhibitor monotherapies.

[0094] The immunocytokine compositions provided herein utilize linkers to attach the antibody or antigen binding fragment to the IL-18 polypeptide. In some embodiments, the linkers are attached to each moiety (i.e., the antibody or antigen binding fragment and the IL-18 polypeptide) at specific residues or a specific subset of residues. In some embodiments, the linkers are attachedWSGR Docket No. 56146-744.601 to each moiety in a site-selective manner, such that a population of the immunocytokine compositions is substantially uniform. This can be accomplished in a variety of ways as provided herein, including by site-selectively adding reagents for a conjugation reaction to a moiety to be conjugated, synthesizing or otherwise preparing a moiety to be conjugated with a desired reagent for a conjugation reaction, or a combination of these two approaches. Using these approaches, the sites of attachment (such as specific amino acid residues) of the linker to each moiety can be selected with precision.

[0095] Additionally, these approaches allow a variety of linkers to be employed for the composition which are not limited to amino acid residues as is required for fusion proteins. This combination of linker choice and precision attachment to the moieties allows the linker to also, in some embodiments, perform the function of modulating the activity of one of the moieties, for example if the linker is attached to the IL-18 polypeptide at a position that interacts with a protein which binds to the IL-18 polypeptide (e.g., IL-18 binding protein).

[0096] In one aspect, provided herein, is an immunocytokine composition, comprising: an IL-18 polypeptide and an antibody or an antigen binding fragment thereof specific for PD-1. In some embodiments, the immunocytokine composition comprises a linker. In some embodiments, the linker comprises a first point of attachment to the IL-18 polypeptide. In some embodiments, the linker comprises a second point of attachmentto the antibody or antigen bindingfragmentthereof.Anti-PD-1 Antibodies

[0097] In some embodiments, an antibody or an antigen binding fragment incorporated into an immunocytokine composition (e.g., by attaching the antibody or antigen binding fragment to an IL-18 polypeptide as described herein) of the disclosure specifically binds to PD-1.

[0098] An antibody selectively binds or preferentially binds to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to specific binding means preferential binding where the affinity of the antibody, or antigen bindingfragmentthereof, is at least at least 2-fold greater, at least 3 -fold greater, at least 4-fold greater, at least 5 -fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the antibody for unrelated amino acid sequences. In some embodiments, an antibody or an antigen bindingfragment of the disclosurecan inhibitthe action / activity of the substanceto which itbinds.WSGR Docket No. 56146-744.601In some embodiments, an antibody or antigen binding fragment of the disclosure can agonize the action / activity of the substance to which it binds (e.g., an immune cell agonist antibody or antigen binding fragment such as one specific for CD16A, NKG2D, NKp30, or other targets).

[0099] As used herein, the term “antibody” refers to an immunoglobulin (Ig), polypeptide, or a protein having a binding domain which is, or is homologous to, an antigen binding domain. The term further includes “antigen binding fragments” and other interchangeable terms for similar binding fragments as described below. Native antibodies and native immunoglobulins (Igs) are generally heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains. Each light chain is typically linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (“VH”) followed by a number of constant domains (“CH”). Each light chain has a variable domain at one end (“VL”) and a constant domain (“CL”) at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.

[0100] In some instances, an antibody or an antigen binding fragment comprises an isolated antibody or antigen binding fragment, a purified antibody or antigen binding fragment, a recombinant antibody or antigen binding fragment, a modified antibody or antigen binding fragment, or a synthetic antibody or antigen binding fragment.

[0101] Antibodies and antigen binding fragments herein can be partly or wholly synthetically produced. An antibody or antigen binding fragment can be a polypeptide or protein having a binding domain which can be, or can be homologous to, an antigen binding domain . In one instance, an antibody or an antigen binding fragment can be produced in an appropriate in vivo animal model and then isolated and / or purified.

[0102] Depending on the amino acid sequence of the constant domain of its heavy chains, immunoglobulins (Igs) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgGl , IgG2, IgG3 , IgG4, IgAl and IgA2. An Ig or portion thereof can, in some cases, be a human Ig. In some instances, a CH3 domain can be from an immunoglobulin. In some cases, a chain or a part of an antibody or antigen binding fragment, a modified antibody or antigen binding fragment, or a binding agent can be from an Ig. In such cases, an Ig can be IgG, an IgA, an IgD, an IgE, or an IgM, or is derived therefrom. In cases where the Ig is an IgG, it canWSGR Docket No. 56146-744.601 be a subtype of IgG, wherein subtypes of IgG can include IgGl, an IgG2a, an IgG2b, an IgG3, or an IgG4. In some cases, a C#3 domain can be from an immunoglobulin selected from the group consisting of an IgG, an IgA, an IgD, an IgE, and an IgM, or is derived therefrom. In some embodiments, an antibody or antigen binding fragment described herein comprises an IgG or is derived therefrom. In some instances, an antibody or antigen binding fragment comprises an IgGl or is derived therefrom. In some instances, an antibody or antigen binding fragment comprises an IgG4 or is derived therefrom. In some instances, an antibody or antigen binding fragment comprises an IgG2 or is derived therefrom. In some embodiments, an antibody or antigen binding fragment described herein comprises an IgM, is derived therefrom, or is a monomeric form of IgM. In some embodiments, an antibody or antigen binding fragment described herein comprises an IgE or is derived therefrom. In some embodiments, an antibody or antigen binding fragment described herein comprises an IgD oris derived therefrom. In some embodiments, an antibody or antigen binding fragment described herein comprises an IgA or is derived therefrom .

[0103] The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (“K” or “K”) or lambda (“X”), based on the amino acid sequences of their constant domains.

[0104] A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed., 1991, National Institutes of Health, Bethesda Md., pages 647-669; hereafter “Kabat”); and (2) an approach based on crystallographic studies of antigen -antibody complexes (Al-Iazikani et al. (1997) J. Molec. Biol. 273 :927-948)). As used herein, a CDRmay refer to CDRs defined by either approach or by a combination of both approaches.

[0105] With respect to antibodies, the term “variable domain” refers to the variable domains of antibodies that are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. Rather, it is concentratedin three segments called hypervariable regions (also known as CDRs) in both the light chain and the heavy chain variable domains. More highly conserved portions of variable domains are called the “framework regions” or “FRs.” The variable domainsWSGR Docket No. 56146-744.601 of unmodified heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4), largely adopting a P-sheet configuration interspersed with three CDRs which form loops connecting and, in some cases, part of the P-sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies see, Kabat).

[0106] The terms “hypervariable region” and “CDR” when used herein, refer to the amino acid residues of an antibody which are responsible for antigen binding. The CDRs comprise amino acid residues from three sequence regions which bind in a complementary manner to an antigen and are known as CDR1, CDR2, and CDR3 for each of the VHand VLchains. In the light chain variable domain, the CDRs typically correspond to approximately residues 24-34 (CDRL1), SO- 56 (CDRL2), and 89-97 (CDRL3), and in the heavy chain variable domain the CDRs typically correspond to approximately residues 31 -35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) according to Kabat et al., Id. It is understood that the CDRs of different antibodies may contain insertions, thus the amino acid numbering may differ. The Kabat numbering system accounts for such insertions with a numbering scheme that utilizes letters attached to specific residues (e.g., 27A, 27B, 27C, 27D, 27E, and 27F of CDRL1 in the light chain) to reflect any insertions in the numberings between different antibodies. Alternatively, in the light chain variable domain, the CDRs typically correspond to approximately residues 26-32 (CDRL1), 50-52 (CDRL2), and 91- 96 (CDRL3), and in the heavy chain variable domain, the CDRs typically correspond to approximately residues 26-32 (CDRH1), 53-55 (CDRH2), and 96-101 (CDRH3) according to Chothia and Lesk (J. Mol. Biol., 196: 901 -917 (1987)).

[0107] As used herein, “framework region,” “FW,” or “FR” refers to framework amino acid residues that form a part of the antigen binding pocket or groove. In some embodiments, the framework residues form a loop that is a part of the antigen binding pocket or groove and the amino acids residues in the loop may or may not contact the antigen. Framework regions generally comprise the regions between the CDRs. In the light chain variable domain, the FRs typically correspond to approximately residues 0-23 (FRL1), 35-49 (FRL2), 57-88 (FRL3), and 98- 109 and in the heavy chain variable domain the FRs typically correspond to approximately residues 0-30 (FRH1), 36-49 (FRH2), 66-94 (FRED), and 103-133 according to Kabat et al., Id. As discussed above with the Kabat numbering for the light chain, the heavy chain too accounts for insertions in a similar manner (e.g., 35 A, 35B ofCDRHl in the heavy chain). Alternatively, in the light chain variable domain, the FRs typically correspond to approximately residues 0-25 (FRL1), 33 9 (FRL2) 53-90 (FRL3), and 97-109 (FRL4), and in the heavy chain variable domain, the FRs typically correspond to approximately residues 0-25 (FRH1), 33-52 (FRH2), 56-95 (FRED), andWSGR Docket No. 56146-744.601102-113 (FRH4) accordingto ChothiaandLesk, / t / . The loop amino acidsof aFR canbe assessed and determinedby inspection of the three-dimensional structureof an antibody heavy chain and / or antibody light chain. The three-dimensional structure canbe analyzed for solvent accessible amino acid positions as such positions are likely to form a loop and / or provide antigen contact in an antibody variable domain. Some of the solvent accessible positions can tolerate amino acid sequence diversity and others (e.g., structural positions) are, generally, less diversified. The three- dimensional structure of the antibody variable domain can be derived from a crystal structure or protein modeling.

[0108] In the present disclosure, the following abbreviations (in the parentheses) are used in accordance with the customs, as necessary: heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (VL), complementarity determining region (CDR), first complementarity determining region (CDR1), second complementarity determining region (CDR2), third complementarity determining region (CDR3), heavy chain first complementarity determining region (VH CDR1), heavy chain second complementarity determining region (VH CDR2), heavy chain third complementarity determining region (VH CDR3), light chain first complementarity determining region (VL CDR1), light chain second complementarity determining region (VL CDR2), and light chain third complementarity determining region (VL CDR3).

[0109] The term “Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is generally defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl -terminus thereof. The numbering of the residues in the Fc region is that of the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3.

[0110] “ Antibodies” useful in the present disclosure encompass, but are not limited to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, bispecific antibodies, grafted antibodies, multispecific antibodies, heteroconjugate antibodies, humanized antibodies, human antibodies, deimmunized antibodies, mutants thereof, fusions thereof, immunoconjugates thereof, antigen binding fragments thereof, and / or any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. In certain embodiments of the methods and conjugates provided herein, the antibody requires an Fc region to enable attachment of a linker between the antibodyWSGR Docket No. 56146-744.601 and the protein (e.g., attachment of the linker using an affinity peptide, such as in AJICAP™ technology).

[0111] In some instances, an antibody is a monoclonal antibody. As used herein, a “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally -occurring mutations that may be present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen (epitope). The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0112] In some instances, an antibody is a humanized antibody. As used herein, “humanized” antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues froma CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and biological activity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences but are included to further refine and optimize antibody performance. In general, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in, for example, WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.

[0113] If needed, an antibody or an antigen bindingfragment describedherein can be assessedfor immunogenicity and, as needed, be deimmunized i.e., the antibody is made less immunoreactive by altering one or more T cell epitopes). As used herein, a “deimmunized antibody” means thatWSGR Docket No. 56146-744.601 one or more T cell epitopes in an antibody sequencehavebeen modified suchthat a T cell response after administration of the antibody to a subject is reduced compared to an antibody that has not been deimmunized. Analysis of immunogenicity and T-cell epitopes present in the antibodies and antigen binding fragments described herein can be carried out via the use of software and specific databases. Exemplary software and databases include iTope™ developed by Antitope of Cambridge, England. iTope™, is an in silico technology for analysis of peptide binding to human MHC class II alleles. The iTope™ software predicts peptide binding to human MHC class II alleles and thereby provides an initial screen for the location of such “potential T cell epitopes.” iTope™ software predicts favorable interactions between amino acid side chains of a peptide and specific binding pockets within the binding grooves of 34 human MHC class II alleles. The location of key binding residues is achieved by the in silico generation of 9mer peptides that overlap by one amino acid spanningthe test antibody variable region sequence. Each 9mer peptide can be tested against each of the 34 MHC class II allotypes and scored based on their potential “fit” and interactions with the MHC class II binding groove. Peptides that produce a high mean binding score (>0.55 in the iTope™ scoring function) against >50% of the MHC class II alleles are considered as potential T cell epitopes. In such regions, the core 9 amino acid sequence for peptide binding within the MHC class II groove is analyzed to determine the MHC class II pocket residues (Pl, P4, P6, P7, and P9) and the possible T cell receptor (TCR) contact residues (P-1, P2, P3, P5, P8). After identification of any T-cell epitopes, amino acid residue changes, substitutions, additions, and / or deletions can be introduced to remove the identified T -cell epitope. Such changes can be made so as to preserve antibody structure and function while still removing the identified epitope. Exemplary changes can include, but are not limited to, conservative amino acid changes.

[0114] An antibody can be a human antibody. As used herein, a “human antibody” means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or thathasbeen made using any suitable technique for making human antibodies. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies. Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Alternatively, the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro).WSGR Docket No. 56146-744.601

[0115] Any of the antibodies herein can be bispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different antigens and can be prepared using the antibodies disclosed herein. Traditionally, the recombinant production of bispecific antibodies was based on the co-expression of two immunoglobulin heavy chain -light chain pairs, with the two heavy chains having different specificities. Bispecific antibodies can be composed of ahybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain -light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure, with an immunoglobulin light chain in only one half of the bispecific molecule, facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations.

[0116] According to one approach to making bispecific antibodies, antibody variable domains with the desired binding specificities (antibody -antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion can be with an immunoglobulin h eavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. The first heavy chain constant region (CHI), containing the site necessary for light chain binding, can be present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

[0117] In some instances, an antibody herein is a chimeric antibody. “Chimeric” forms of nonhuman (e.g., murine) antibodies include chimeric antibodies which contain minimal sequence derived from a non-human Ig. For the most part, chimeric antibodies are murine antibodies in which at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, is inserted in place of the murine Fc. Chimeric or hybrid antibodies also may be prepared in vitro using suitable methods of synthetic protein chemistry, including those involving cross-linking agents.

[0118] Provided herein are antibodies and antigen bindingfragments thereof, modified antibodies and antigen binding fragments thereof, and binding agents that specifically bind to one or more epitopes on one or more target antigens. In one instance, a binding agent selectively binds to an epitope on a single antigen. In another instance, a binding agent is bivalent and either selectivelyWSGR Docket No. 56146-744.601 binds to two distinct epitopes on a single antigen or binds to two distinct epitopes on two distinct antigens. In another instance, a binding agent is multivalent (z.e., trivalent, quatravalent, etc. and the binding agent binds to three or more distinct epitopes on a single antigen or binds to three or more distinct epitopes on two or more (multiple) antigens.

[0119] Antigen binding fragments of any of the antibodies herein are also contemplated. The terms “antigen binding portion of an antibody,” “antigen binding domain,” “antibody fragment,” or a “functional fragment of an antibody” are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Representative antigen binding fragments include, but are not limited to, a Fab, a Fab', a F(ab')2, a bispecific F(ab')2, a trispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a dsFv, a bispecific scFv, a variable heavy domain, a variable light domain, a variable NAR domain, bispecific scFv, an AVIMER®, a minibody, a diabody, abispecific diabody,triabody, a tetrabody, a minibody, a maxibody, a camelid, a VHH, a minibody, an intrabody, fusion proteins comprising an antibody portion (e.g., a domain antibody), a single chain binding polypeptide, a scFv-Fc, a Fab-Fc, a bispecific T cell engager (BiTE; two scFvs produced as a single polypeptide chain, where each scFv comprises an amino acid sequences a combination of CDRs or a combination of VL / VL described herein), a tetravalent tandem diabody (TandAb; an antibody fragment that is produced as a non-covalent homodimer folder in a head-to-tail arrangement, e.g., a TandAb comprising an scFv, where the scFv comprises an amino acid sequences a combination of CDRs or a combination of VL / VL described herein), a Dual-Affinity Re-targeting Antibody (DART; different scFvs joined by a stabilizing interchain disulphide bond), a bispecific antibody (bscAb; two single-chain Fv fragments joined via a glycine -serine linker), a single domain antibody (sdAb), a fusion protein, a bispecific disulfide-stabilized Fv antibody fragment (dsFv-dsFv'; two different disulfide-stabilized Fv antibody fragments connected by flexible linker peptides). In certain embodiments of the invention, a full length antibody (e.g., an antigen binding fragment and an Fc region) are preferred.

[0120] Heteroconjugate polypeptides comprising two covalently joined antibodies or antigen binding fragments of antibodies are also within the scope of the disclosure. Suitable linkers may be used to multimerize binding agents. Non-limiting examples of linking peptides include, but are not limited to, (GS)n(SEQ ID NO: 124), (GGS)n(SEQ ID NO: 125), (GGGS)n(SEQ ID NO: 126), (GGSG)n(SEQ ID NO: 127), or (GGSGG)n(SEQ ID NO: 128), (GGGGS)n(SEQ ID NO: 129), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, a linking peptide can be (GGGGS)3(SEQ ID NO: 130) or (GGGGS)4 (SEQ ID NO: 123). In some embodiments, a linking peptide bridges approximately 3.5 nm between the carboxy terminus of one variable region and the aminoWSGR Docket No. 56146-744.601 terminus of the other variable region. Linkers of other sequences have been designed and used. Linkers can in turn be modified for additional functions, such as attachment of drugs or attachment to solid supports.

[0121] As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution. Apparent affinities can be determined by methods such as an enzyme-linked immunosorbent assay (ELISA) or any other suitable technique. Avidities can be determined by methods such as a Scatchard analysis or any other suitable technique.

[0122] As used herein, the term “affinity” refers to the equilibrium constant for the reversible binding of two agents and is expressed as KD. The binding affinity (KD) of an antibody or antigen binding fragment herein can be less than 500 nM, 475 nM, 450 nM, 425 nM, 400 nM, 375 nM, 350 nM, 325 nM, 300 nM, 275 nM, 250 nM, 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, 100 nM, 90 nM, 80 nM, 70 nM, 50 nM, 50 nM, 49 nM, 48 nM, 47 nM, 46 nM, 45 nM, 44 nM, 43 nM, 42 nM, 41 nM, 40 nM, 39 nM, 38 nM, 37 nM, 36 nM, 35 nM, 34 nM, 33 nM, 32 nM, 31 nM, 30 nM, 29 nM, 28 nM, 27 nM, 26 nM, 25 nM, 24 nM, 23 nM, 22 nM, 21 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, or any integer therebetween. Binding affinity may be determined using surface plasmon resonance (SPR), KINEXA® Biosensor, scintillation proximity assays, enzyme linked immunosorbent assay (ELISA), ORIGEN immunoassay (IGEN), fluorescence quenching fluorescence transfer, yeast display, or any combination thereof. Binding affinity may also be screened using a suitable bioassay.

[0123] Also provided herein are affinity matured antibodies. The following methods may be used for adjusting the affinity of an antibody and for characterizing a CDR. One way of characterizing a CDR of an antibody and / or altering (such as improving) the binding affinity of a polypeptide, such as an antibody, is termed “library scanning mutagenesis.” Generally, library scanning mutagenesis works as follows. One or more amino acid position in the CDR is replaced with two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids. ThisWSGR Docket No. 56146-744.601 generates small libraries of clones (in some embodiments, one for every amino acid position that is analyzed), each with a complexity of two or more members (if two or more amino acids are substituted at every position). Generally, the library also includes a clone comprising the native (unsubstituted) amino acid. A small number of clones, for example, about 20-80 clones (depending on the complexity of the library), from each library can be screened for binding specificity or affinity to the target polypeptide (or other binding target), and candidates with increased, the same, decreased, or no binding are identified. Binding affinity may be determined using Biacore surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or greater.

[0124] In some instances, an antibody or antigen binding fragment is bispecific or multispecific and can specifically bind to more than one antigen. In some cases, such a bispecific or multispecific antibody or antigen binding fragment can specifically bind to 2 or more different antigens. In some cases, a bispecific antibody or antigen binding fragment can be a bivalent antibody or antigen binding fragment. In some cases, a multi specific antibody or antigen binding fragment can be a bivalent antibody or antigen binding fragment, a trivalent antibody or antigen binding fragment, or a quatravalent antibody or antigen binding fragment.

[0125] An antibody or antigen binding fragment described herein can be isolated, purified, recombinant, or synthetic.

[0126] It is contemplated that generic or biosimilar versions of the named antibodies herein which share the same amino acid sequence as the indicated antibodies are also encompassed when the name of the antibody is used.

[0127] The antibodies described herein may be made by any suitable method. Antibodies can often be produced in large quantities, particularly when utilizing high level expression vectors.

[0128] In one embodiment, an antibody or an antigen binding fragment of the disclosure comprises a fusion protein or a peptide immunotherapeutic agent.

[0129] In some embodiments, the antibody or antigen binding fragment thereof of an immunocytokine composition descried herein is an anti-PD-1 antibody or antigen binding fragment. Programmed cell death protein 1 (also known as PD-1 and CD279), is a cell surface receptor that plays a role in down -regulating the immune system and promoting self-tolerance by suppressing cell inflammatory activity. PD-1 is an immune cell inhibitory molecule that is expressed on activatedB cells, T cells, and myeloid cells. PD-1 represents an immune checkpoint and guards against autoimmunity via a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T-cells in lymph nodes while reducing apoptosis in regulatory T cells. PD-1 is a member of the CD28 / CTLA-4 / ICOS costimulatory receptor family that deliversWSGR Docket No. 56146-744.601 negative signals that affect T and B cell immunity. PD-1 is monomeric both in solution as well as on cell surface, in contrast to CTLA-4 and other family members that are all disulfide-linked homodimers. Signaling through the PD-1 inhibitory receptor upon binding its ligand, PD-L1, suppresses immuneresponses again st autoantigens and tumors andplay s a role in the maintenance of peripheral immune tolerance. The interaction betweenPD-1 and PD-L1 results in a decrease in tumor infiltrating lymphocytes, a decrease in T cell receptor mediated proliferation, and immune evasion by the cancerous cells. A non-limiting, exemplary, human PD-1 amino acid sequence is MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSN TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN DSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 131).

[0130] In one embodiment, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment of the disclosure comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) described herein. In another embodiment, an anti-PD-1 antibody or an anti- PD-1 antigen binding fragment of the disclosure comprises a combination of complementarity determining regions (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) described herein. In one embodiment, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment of the disclosure comprises a modified Tislelizumab, Baizean, 0KVO411B3N, BGB- A317, hu317-l / IgG4mt2, Sintilimab, Tyvyt, IBI-308, Toripalimab, TeRuiPuLi, Terepril, Tuoyi, JS-001, TAB-001, Camrelizumab, HR-301210, INCSHR-01210, SHR-1210, Cemiplimab, Cemiplimab-rwlc, LIBTAYO®, 6QVL057INT, H4H7798N, REGN-2810, SAR-439684, Avelumab, BAVENCIO®, 451238, KXG2PJ551I, MSB-0010682, MSB-0010718C, PF- 06834635, Durvalumab, IMFINZI®, 28X28X9OKV, MEDI-4736, Lambrolizumab, Pembrolizumab, KEYTRUDA®, MK-3475, SCH-900475, h409Al l, Nivolumab, Nivolumab BMS, OPDIVO®, BMS-936558, MDX-1106, ONO-4538, Prolgolimab, Forteca, BCD-100, Penpulimab, AK-105, Zimberelimab, AB-122, GLS-010, WBP-3055, Balstilimab, 1Q2QT5M7EO, AGEN-2034, AGEN-2034w, Genolimzumab, Geptanolimab, APL-501, CBT- 501, GB-226, Dostarlimab, ANB-011, GSK-4057190A, P0GVQ9A4S5, TSR-042, WBP-285, Serplulimab, HLX-10, CS-1003, Retifanlimab, 2Y3T5IF01Z, INCMGA-00012, INCMGA-0012, MGA-012, Sasanlimab, LZZ0IC2EWP, PF-06801591, RN-888, Spartalizumab,NVP-LZV-184, PDR-001, QOG25L6Z8Z, Relatlimab / nivolumab, BMS-986213, Cetrelimab, JNJ-3283, JNJ- 63723283, LYK98WP91F, Tebotelimab, MGD-013, BCD-217, BAT-1306, HX-008, MEDLWSGR Docket No. 56146-744.6015752, JTX-4014, Cadonilimab, AK-104, BL754091, Pidilizumab, CT-011, MDV-9300, YBL- 006, AMG-256, RG-6279, RO-7284755, BH-2950, IBI-315, RG-6139, RO-7247669, ONO 685, AK-112, 609-A, LY-3434172, T-3011, MAX-10181, AMG-404, IBI-318, MGD-019, INCB- 086550, ONCR-177,LY-3462817, RG-7769, RO-7121661, F-520, XmAb-23104, Pd-l-pik, SG- 001, S-95016, Sym-021, LZM-009 (a.k.a., Lipustobart), Budigalimab, 6VDO4TY3OO, ABBV- 181 , PR-1648817, CC-90006, XmAb-20717, 2661380, AMP-224, B7-DCIg, EMB-02, ANB-030, PRS-332, [89Zr]Deferoxamide-pembrolizumab, 89Zr-Df-Pembrolizumab, [89Zr]Df- Pembrolizumab, STI-1110, STI-Al l 10, CX-188, mPD-1 Pb-Tx, MCLA-134, 244C8, ENUM 224C8, ENUM C8, 388D4, ENUM 388D4, ENUM D4, MEDI0680, or AMP-514.

[0131] In some embodiments, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment of the disclosure comprises a Tislelizumab, Sintilimab, Toripalimab, Terepril, Camrelizumab, Cemiplimab, Pembrolizumab Nivolumab, Prolgolimab, Penpulimab, Zimberelimab, Balstilimab, Genolimzumab, Geptanolimab, Dostarlimab, Serplulimab, Retifanlimab, Sasanlimab, Spartalizumab, Cetrelimab, Tebotelimab, Cadonilimab, Pidilizumab, LZM-009 (a.k.a. Lipustobart), or Budigalimab. In one embodiment, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment of the disclosure comprises a modified Tislelizumab, Sintilimab, Toripalimab, Terepril, Camrelizumab, Cemiplimab, Pembrolizumab Nivolumab, Prolgolimab, Penpulimab, Zimberelimab, Balstilimab, Genolimzumab, Geptanolimab, Dostarlimab, Serplulimab, Retifanlimab, Sasanlimab, Spartalizumab, Cetrelimab, Tebotelimab, Cadonilimab, A Pidilizumab, LZM-009 (a.k.a. Lipustobart), or Budigalimab.

[0132] In some embodiments, the anti-PD-1 polypeptide is Nivolumab, Pembrolizumab, LZM- 009, Dostarlimab, Sintilimab, Spartalizumab, Tislelizumab, orCemiplimab. In some embodiment, the anti-PD-1 polypeptide is Dostarlimab, Sintilimab, Spartalizumab, or Tislelizumab. In some embodiments, the anti-PD-1 polypeptide is Nivolumab, Pembrolizumab, LZM-009 (a.k.a. Lipustobart), or Cemiplimab.

[0133] In some embodiments, the anti-PD-1 antibody is Pembrolizumab. In some embodiments, the anti-PD-1 antibody is modified Pembrolizumab.

[0134] In some preferred embodiments, the anti-PD-1 antibody is LZM-009 (Lipustobart). In some embodiments, the anti-PD-1 antibody is modified LZM-009. LZM-009 (a.k.a. Lipustobart) has a full-length light chain sequence ofEIVLTQSPATLSLSPGERATISCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSRELPLTFGTGTKVEIKRTVAAPSVFIF PPSDEQLKSGTAS VVCLLNNF YPREAK VQWKVDNALQ SGNSQES VTEQD SKD STYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 73) and a full-lengthWSGR Docket No. 56146-744.601 heavy chain sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWMGGVNPSNG GTNFNEKFKSRVTITADKSTSTAYMELSSLRSEDTAVYYCARRDYRYDMGFDYWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 74).

[0135] In some embodiments, the anti-PD-1 antibody is a biosimilar of Tislelizumab, Sintilimab, Toripalimab, Terepril, Camrelizumab, Cemiplimab, Pembrolizumab Nivolumab, Prolgolimab, Penpulimab, Zimberelimab, Balstilimab, Genolimzumab, Geptanolimab, Dostarlimab, Serplulimab, Retifanlimab, Sasanlimab, Spartalizumab, Cetrelimab, Tebotelimab, Cadonilimab, A Pidilizumab, LZM-009, or Budigalimab. In some embodiments, the anti-PD-1 antibody is a biosimilar of any one of the antibodies provided herein.

[0136] TABLE 1 provides the sequences of exemplary anti-PD-1 antibodies and anti-PD-1 antigen binding fragments that can be modified to prepare anti-PD-1 immunoconjugates. TABLE 1 also shows provides combinations of CDRs that can be utilized in a modified anti-PD-1 immunoconjugate. Reference to an anti-PD-1 antibody herein may alternatively refer to an anti- PD-1 antigen binding fragment.

[0137] In some instances, the SEQ ID NOs listed in Table 1 contain full-length heavy or light chains of the indicated antibodies with the VH or VL respectively indicated in bold. Where there is a reference herein to a VH or VL of a SEQ ID NO in Table 1 which contains a full-length heavy orlight chain, itis intended to referencethe bolded portion of the sequence. For example, reference to “a VH having an amino acid sequence shown in SEQ ID NO: 132” refers to the bolded portion of SEQ ID NO: 132 in Table 1.

[0138] An anti-PD-1 antibody or an anti-PD-1 antigen binding fragment can comprise a VH having an amino acid sequence of any one of SEQ ID NOS: 132, 134, 136, 138, 140, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, and 178. An anti-PD-1 antibody or an anti-PD-1 antigen binding fragment can comprise a VL having an amino acid sequence ofany one of SEQ ID NOS: 133, 135, 137, 139, 141, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, and 179.

[0139] An anti-PD-1 antibody or an anti-PD-1 antigen binding fragment can comprise a heavy chain or VH having an amino acid sequence of any one of SEQ ID NOS: 132, 134, 136, 138, 140,WSGR Docket No. 56146-744.601146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, and 178, or a portion corresponding to a VH thereof. An anti-PD-1 antibody or an anti-PD-1 antigen binding fragment can comprise a light chain or VL having an amino acid sequence of any one of SEQ ID NOS: 133, 135, 137, 139, 141, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, and 179, or a portion corresponding to a VL thereof.

[0140] In one instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 132, and a VL having an amino acid sequence shown in SEQ ID NO: 133. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 134, and a VL having an amino acid sequence shown in SEQ ID NO: 135. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 136, and a VL having an amino acid sequence shown in SEQ ID NO: 137. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen bindingfragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 138, and a VL having an amino acid sequence shown in SEQ ID NO: 139. In another instance, an anti-PD- 1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 140, and a VL having an amino acid sequence shown in SEQ ID NO: 141. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 146, and a VL having an amino acid sequence shown in SEQ ID NO: 147. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 148, and aVL havingan amino acid sequence shownin SEQ ID NO: 149. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 150, and a VL having an amino acid sequence shown in SEQ ID NO: 151. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen bindingfragment comprises a VH having an amino acid sequence shownin SEQ ID NO: 152, and a VL having an amino acid sequence shown in SEQ ID NO: 153. In another instance, an anti-PD- 1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 154, and a VL having an amino acid sequence shown in SEQ ID NO: 155. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 156, and a VL having an amino acid sequence shown in SEQ ID NO: 157. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 158, and a VL havingan amino acid sequence shownin SEQ ID NO: 159. In anotherWSGR Docket No. 56146-744.601 instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 160, and a VL having an amino acid sequence shown in SEQ ID NO: 161. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen bindingfragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 162, and a VL having an amino acid sequence shown in SEQ ID NO: 163. In another instance, an anti-PD- 1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 164, and a VL having an amino acid sequence shown in SEQ ID NO: 165. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 166, and a VL having an amino acid sequence shown in SEQ ID NO: 167. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 168, and aVL having an amino acid sequence shown in SEQ ID NO: 169. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 170, and a VL having an amino acid sequence shown in SEQ ID NO: 171. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen bindingfragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 172, and a VL having an amino acid sequence shown in SEQ ID NO: 173. In another instance, an anti-PD- 1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 174, and a VL having an amino acid sequence shown in SEQ ID NO: 175. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 176, and a VL having an amino acid sequence shown in SEQ ID NO: 177. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence shown in SEQ ID NO: 178, and a VL having an amino acid sequence shown in SEQ ID NO: 179.

[0141] In one instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 132, and a VL having an amino acid sequence of SEQ ID NO: 133. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 134, and a VL having an amino acid sequence of SEQ ID NO: 135. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 136, and a VL having an amino acid sequence of SEQ ID NO: 137. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 138, and a VL having an amino acid sequence of SEQ ID NO: 139. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen bindingWSGR Docket No. 56146-744.601 fragment comprises a VH having an amino acid sequence of SEQ ID NO: 140, and a VL having an amino acid sequence of SEQ ID NO: 141. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 146, and a VL having an amino acid sequence of SEQ ID NO: 147. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 148, and a VL having an amino acid sequence of SEQ ID NO: 149. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 150, and a VL having an amino acid sequence of SEQ ID NO: 151 . In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 152, and a VL having an amino acid sequence of SEQ ID NO: 153. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 154, and a VL having an amino acid sequence of SEQ ID NO: 155. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 156, and a VL having an amino acid sequence of SEQ ID NO: 157. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 158, and a VL having an amino acid sequence of SEQ ID NO: 159. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 160, and a VL having an amino acid sequence of SEQ ID NO: 161. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 162, and a VL having an amino acid sequence of SEQ ID NO: 163. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 164, and a VL having an amino acid sequence of SEQ ID NO: 165. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 166, and a VL having an amino acid sequence of SEQ ID NO: 167. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 168, and a VL having an amino acid sequence of SEQ ID NO: 169. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 170, and a VL having an amino acid sequence of SEQ ID NO: 171. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 172, and a VL having an amino acid sequence of SEQ ID NO: 173. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises aWSGR Docket No. 56146-744.601VH having an amino acid sequence of SEQ ID NO: 174, and a VL having an amino acid sequence of SEQ ID NO: 175. In another instance, an anti-PD-1 antibody or an anti -PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 176, and a VL having an amino acid sequence of SEQ ID NO: 177. In another instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH having an amino acid sequence of SEQ ID NO: 178, and a VL having an amino acid sequence of SEQ ID NO: 179.

[0142] In one instance, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 180, a VH CDR2 having an amino acid sequence of SEQ ID NO: 181 , a VH CDR3 having an amino acid sequence of SEQ ID NO: 182, VL CDR1 having an amino acid sequence of SEQ ID NO: 183, a VL CDR2 having an amino acid sequence of SEQ ID NO: 184, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 185. In one instance, an anti-PD-1 antibody or an anti-PD-1 antigen bindingfragment comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 186, a VH CDR2 having an amino acid sequence of SEQ ID NO: 187, a VH CDR3 having an amino acid sequence of SEQ ID NO: 188, VL CDR1 having an amino acid sequence of SEQ ID NO: 189, a VL CDR2 having an amino acid sequence of SEQ ID NO: 190, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 191. In one instance, an anti-PD-1 antibody or an anti-PD-1 antigen bindingfragment comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 192, a VH CDR2 having an amino acid sequence of SEQ ID NO : 193 , a VH CDR3 having an amino acid sequence of SEQ ID NO: 194, VL CDR1 having an amino acid sequence of SEQ ID NO: 195, a VL CDR2 having an amino acid sequence of SEQ ID NO: 196, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 197. In one instance, an anti-PD-1 antibody or an anti-PD-1 antigen bindingfragment comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 198, a VH CDR2 having an amino acid sequence of SEQ ID NO: 199, a VH CDR3 having an amino acid sequence of SEQ ID NO: 100, VL CDR1 having an amino acid sequence of SEQ ID NO: 101, a VL CDR2 having an amino acid sequence of SEQ ID NO: 102, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 103.

[0143] In oneinstance, an anti-PD-1 antibody comprises afusion protein. Such fusion protein can be, for example, a two-sided Fc fusion protein comprising the extracellular domain (ECD) of programmed cell death 1 (PD-1) and the ECD of tumor necrosis factor (ligand) superfamily member 4 (TNFSF4 or OX40L) fused via hinge-CH2-CH3 Fc domain of human IgG4, expressed in CHO-K 1 cells, where the fusion protein has an exemplary amino acid sequence of SEQ ID NO : 104.WSGR Docket No. 56146-744.601TABLE 1 - Exemplary Antibodies for Immune Cell Associated AntigensWSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601Modification to Fc region

[0144] In some embodiments, the antibodies or antigen binding fragments thereof incorporated into immunocytokine compositions described herein comprise an Fc region, wherein the Fc regionWSGR Docket No. 56146-744.601 comprises at least one covalently linked linker. In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is covalently attached to a tyrosine, aspartic acid, glutamic acid, arginine, histidine, or lysine residue. In some embodiments, the chemical linker is covalently attached to a lysine, cysteine, or tyrosine residue. In some embodiments, the chemical linker is covalently attached to a cysteine residue. In some embodiments, the chemical linker is covalently attached to a lysine residue. In some embodiments, the chemical linker is covalently attached to a constant region of the antibody.

[0145] In some embodiments, the antibody of the immunocytokine composition comprises anFc region. In some embodiments, the Fc region is an IgG Fc region, an IgA Fc region, an IgD Fc region, an IgM Fc region, or an IgE Fc region. In some embodiments, the Fc region is an IgG Fc region, an IgA Fc region, or an IgD Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, theFc region is a humanized. Fc region. In some embodiments, the Fc region is an IgG Fc region. In some instances, anlgGFc region is an IgGl Fc region, an IgG2a Fc region, or an IgG4 Fc region.

[0146] One or more mutations may be introduced in an Fc region to reduce Fc -mediated effector functions of an antibody or antigen -binding fragment such as, for example, antibody -dependent cellular cytotoxicity (ADCC) and / or complement function. In some instances, a modified Fc comprises a humanized IgG4 kappa isotype that contains a S229P Fc mutation. In some instances, a modified Fc comprises a human IgGl kappa where the heavy chain CH2 domain is engineered with a triple mutation such as, for example: (a) L238P, L239E, and P335S; or (2) K248; K288; and K317.

[0147] In some embodiments, the Fc region has an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence as set forth in SEQ ID NO: 80 (Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Xaa Xaa Gly Xaa Pro Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asp Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys Asn Gin Vai Ser Leu Thr Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp He Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Xaa Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys SerWSGR Docket No. 56146-744.601Leu Ser Leu Ser Pro Gly, where Xaa can be any naturally occurring amino acid) . In some embodiments, the Fc region comprises one or more mutations which make the Fc region susceptible to modification or conjugation at a particular residue, such as by incorporation of a cysteine residue at a position which does not contain a cysteine in SEQ ID NO: 80. Alternatively, the Fc region could be modified to incorporate a modified natural amino acid or an unnatural amino acid which comprises a conjugation handle, such as one connected to the modified natural amino acid or unnatural amino acid through a linker. In some embodiments, the Fc region does not comprise any mutations which facilitate the attachment of a linker to an additional cytokine (e.g., an IL-18 polypeptide). In some embodiments, the chemical linker is attached to a native residue as set forth in SEQ ID NO: 80. In some embodiments, the chemical linker is attached to a native lysine residue of SEQ ID NO: 80.

[0148] In some embodiments, the chemical linker is attached to the Fc region at an amino acid residue at one of positions 10-90 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at an amino acid residue at one of positions 10 -20, 10-30, 10-40, 10- 50, 10-60, 10-70, 1-80, 10-90, 10-100, 10-110, 10-120, 10-130, 10-140, 10-150, 10-160, 10-170, 10-180, 10-190, or 10-200 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at an amino acid residue at one of positions 10-30, 50-70, or 80-100 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at an amino acid residue at any one of positions 20-40, 65-85, or 90-110 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at an amino acid residue at one of positions 15-26, 55-65, or 85-90 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at an amino acid residue at any one of positions 25 -35, 70-80, or 95-105 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at an amino acid residue at any one of positions 30, 32, 72, 74, or 101 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at an amino acid residue at any one of positionsK30, K32,K72,K74,orK101 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 30 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 32 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 72 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 74 of SEQ ID NO: 80. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 101 of SEQ ID NO: 80.

[0149] The chemical linker can be covalently attached to one amino acid residue of an Fc region of the antibody. In some embodiments, the chemical linker is covalently attached to a non -terminalWSGR Docket No. 56146-744.601 residue of the Fc region. In some embodiments, the non-terminal residue is in the CHI, CH2, or CH3 region of the antibody. In some embodiments, the non-terminal residue is in the CH2 region of the antibody.

[0150] In some embodiments, the chemical linker is covalently attached at an amino acid residue of the antibody orantigenbindingfragmentwhich selectivelybinds an immune associated antigen (e.g., an anti-PD-1 antibody) such that the function of the antibody or antigen binding fragment is maintained (e.g., without denaturing the polypeptide). For example, when the antibody or antigen binding fragment is a human IgG (e.g., human IgGl), exposed lysine residues and exposed tyrosine residues are present at the following positions (refer to web site www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html by EU numbering). Exemplary exposed Lysine Residues: CH2 domain (position 246, position 248, position 274, position 288, position 290, position 317, position 320, position 322, and position 338) CH3 domain (position 360, position 414, and position 439). Exemplary exposed Tyrosine Residues: CH2 domain (position 278, position 296, and position 300) CH3 domain (position 436).

[0151] The human IgG, such as human IgGl, may also be modified with a lysine or tyrosine residue at any one of the positions listed above in order provide a residue which is ideally surface exposed for subsequent modification.

[0152] In some embodiments, the chemical linker is covalently attached at an amino acid residue in the constant region of an antibody. In some embodiments, the chemical linker is covalently attached at an amino acid residue in the CHI, CH2, or CH3 region. In some embodiments, the chemical inker is covalently attached at an amino acid residue in the CH2 region. In some embodiments, the chemical linker may be covalently attached to one amino acid residue in the following groups of residues following EU numbering in human IgG Fc: amino acid residues 1 - 478, amino acid residues 2-478, amino acid residues 1 -477, amino acid residues 2-477, amino acid residues 10-467, amino acid residues 30-447, amino acid residues 50-427, amino acid residues 100-377, amino acid residues 150-327, aminoacid residues 200-327, amino acid residues 240-327, and amino acid residues 240-320.

[0153] In some embodiments, the chemical linker is covalently attached to one lysine residue of a human IgGFc region. In some embodiments, the chemical linker is covalently attached atLys 246, Lys 248, Lys 288, Lys 290, or Lys 317 of the Fc region (EU numbering). In some embodiments, the chemical linker is covalently attached at Lys 246 of an Fc region of the antibody, wherein amino acid residue position number is based on EU numbering. In some embodiments, the chemical linker is covalently attached at Lys 248 of an Fc region of the antibody, wherein amino acid residue position number is based on EU numbering. In someWSGR Docket No. 56146-744.601 embodiments, the chemical linker is covalently attached at Lys 288 of an Fc region of the antibody, wherein amino acid residue position number is based on EU numbering. In some embodiments, the chemical linker is covalently attached at Lys 290 of an Fc region of the antibody, wherein amino acid residue position number is based on EU numbering. In some embodiments, the chemical linker is covalently attached at Lys 317 of the antibody, wherein amino acid residue position number is based on EU numbering.

[0154] The chemical linker can be covalently attached to an amino acid residue selected from a subset of amino acid residues. In some embodiments, the subset comprises two three, four, five, six, seven, eight, nine, or ten amino acid residues of an Fc region of the antibody. The chemical linker can be covalently attached to one of two lysine residues of an Fc region of the antibody.

[0155] In some embodiments, the antibody will comprise two linkers covalently attached to the Fc region of the antibody. In some embodiments, each of the two linkers will be covalently attached to a different heavy chain of the antibody. In some embodiments, each of the two linkers will be covalently attached to a different heavy chain of the antibody at a residue position which is the same. In some embodiments, each of the two linkers will be covalently attached to a different heavy chain of antibody at a residue position which is different. When the two linkers are covalently attached to residue positions which differ, any combination of the residue positions provided herein may be used in combination. However, in preferred embodiments of immunocytokine compositions described herein, only a single linker is attached to the antibody or antigen binding fragment thereof, thereby providing an immunocytokine composition which includes only a single IL-18 polypeptide described herein (e.g., a single IL-18 polypeptide of SEQ ID NO: 30, for example).Method of Modifying an Fc Region

[0156] Also provided herein are method of preparing a modified Fc region of an antibody or antigen binding fragment, such as for the attachment of a linker, a conjugation handle, the IL-18 polypeptide, or any combination thereof to the antibody or antigen binding fragment to form the immunocytokine composition. A variety of methods for site-specific modification of Fc regions of antibodies are known in the art.Modification with an affinity peptide configured to site -specifically attach linker to the antibody

[0157] In some embodiments, an Fc region is modified to incorporate a linker, a conjugation handle, or a combination thereof. In some embodiments, the modification is performed by contacting the Fc region with an affinity peptide bearing a payload configured to attach a linker or other group to the Fc region, such as at a specific residue of the Fc region. In some embodiments, the linker is attached using a reactive group which forms a bond with a residue ofWSGR Docket No. 56146-744.601 the Fc region. In some embodiments, the affinity peptide comprises a cleavable linker. The cleavable linker is configured on the affinity peptide such that after the linker or other group is attached to the Fc region, the affinity peptide can be removed, leavingbehind only the desired linker or other group attached to the Fc region. The linker or other group can then be used further to add attach additional groups, such as a cytokine or a linker attached to a cytokine, to the Fc region.

[0158] Non-limiting examples of such affinity peptides can be found at least in PCT Publication No. WO2018199337A1, PCT Publication No. WO2019240288A1, PCT Publication No. WO2019240287A1, and PCT Publication No. W02020090979A1, each of which is incorporated by reference as if set forth herein in its entirety. In some embodiments, the affinity peptide is a peptide which has been modified to deliver the linker / conjugation handle payload one or more specific residues of the Fc region of the antibody. In some embodiments, the affinity peptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identify to a peptide selected from (1)QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 81); (2)QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQ APQA (SEQ ID NO: 82); (3) QETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 83); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 84); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDDC (SEQ ID NO: 85); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 86); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 87); (8)QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 88); (9)QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 89); (10)QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 90); and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 91), or a corresponding peptide which has been truncated at the N-terminus by one, two, three, four, or five residues.

[0159] An exemplary affinity peptide with cleavable linker and conjugation handle payload capable of attaching the payload to residueK248 of an antibody as provided herein is shown below (SEQ ID NO: 92) (as reported in Matsuda et al. , “Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody -Drug Conjugates,” Mol Pharmaceutics 2021, 18, 11, 4058-4066.WSGR Docket No. 56146-744.601

[0160] Alternative affinity peptides targeting alternative residues of the Fc region are described in the references cited above for AJICAP™ technology, and such affinity peptides can be used to attach the desired functionality to an alternative residue of the Fc region (e.g., K246, K288, etc.). For example, the disulfide group of the above affinity peptide could instead be replaced with a thioester to provide a sulfhydryl protecting group as a cleavable portion of the linking group (e.g, the relevant portion of the affinity peptide would have a structure ofor another of the cleavable linkers discussed below). Such alternative affinity peptides includethose described in, for example “ AJICAP Second Generation:Improved Chemical Site-Specific Conjugation Technology for Antibody -Drug ConjugationTechnology for Antibody -Drug Conjugate Production” (Working Paper, Fujii et al., DOI: 10.26434 / chemrxiv-2023-9p5p7, chemrxiv.org / engage / chemrxiv / article- details / 63d5f7131125965a9e7df8a5 (Accessed 20 February 2023, Version 1 published 30 Jan 2023)). Exemplary affinity peptides provided therein include those shown below, wherein the left structure (SEQ ID NO: 93) targets K248 of the Fc region and the right structure (SEQ ID NO: 94) targets K288 of the Fc region (EU numbering).

[0161] The affinity peptide of the disclosure can comprise a cleavable linker. In some embodiments, the cleavable linker of the affinity peptide connects the affinity peptide to the group which is to be attached to the Fc region and is configured such that the peptide can be cleavedWSGR Docket No. 56146-744.601 after the group comprising the linker or conjugation handle has been attached. In some embodiments, the cleavable linker is a divalent group. In some embodiments, the cleavable linker can comprise a thioester group, an ester group, a sulfane group; a methanimine group; an oxy vinyl group; a thiopropan oate group; an ethane- 1 ,2-diol group; an (imidazole- 1 -yljmethan- 1-one group; a seleno ether group; a silylether group; a di-oxysilane group; an ether group; a di-oxymethane group; a tetraoxospiro[5.5]undecane group; an acetamidoethyl phosphoramidite group; a bis(methylthio)-pyrazolopyrazole-dione group; a 2-oxo-2 -phenylethyl formate group; a 4- oxybenzylcarbamate group; a 2-(4-hydroxy-oxyphenyl)diazinyl)benzoic acid group; a 4-amino- 2-(2-amino-2-oxoethyl)-4-oxobut-2-enoic acid group; a 2-(2-methylenehydrazineyl)pyridine group; an N'-methyleneformohydrazide group; or an isopropylcarbamate group, any of which is unsubstituted or substituted. Composition and points of attachment of the cleavable linker to the affinity peptide, as well as related methods of use, are described in, at least, PCT Publication No. WO2018199337A1, PCT Publication No. WO2019240288A1, PCT Publication No. WO20 19240287 Al, and PCT Publication No. W02020090979A1.

[0162] In some embodiments, the cleavable linker is:WSGR Docket No. 56146-744.601wherein:-one of A or B is a point of attachment the linker and the other of A or B is a point of attachment to the affinity peptide;- each R2ais independently H or optionally substituted alkyl;- each R2bis independently H or optionally substituted alkyl;- R2cis a H or optionally substituted alkyl;-J is a methyl, a N, a S, a Si, or an O atom; and- r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0163] The affinity peptide comprises a reactive group which is configured to enable the covalent attachment of the linker / conjugation handle to the Fc region. In some embodiments, the reactive group is selective for a functional group of a specific amino acid residue, such as a lysine residue, tyrosine residue, serine residue, cysteine residue, or an unnatural amino acid residue of the Fc region incorporated to facilitate the attachment of the linker. The reactive group may be any suitable functional group, such as an activated ester for reaction with a lysine (e.g., billy droxy succinimide ester or a derivate thereof, a pentafluorophenyl ester, etc.) or a sulfhydryl reactive group for reaction with a cysteine (e.g., a Michael acceptor, such as an alpha-beta unsaturated carbonyl or a maleimide). In some embodiments, the reactive group is:WSGR Docket No. 56146-744.601, wherein:- each R5a, R5b, and R5cis independently H, halogen, or optionally substituted alkyl;- each j is 1, 2, 3, 4, or 5; and- each k is 1, 2, 3, 4, or 5.

[0164] In some embodiments, the affinity peptide is used to deliver a reactive moiety to the desired amino acid residue such thatthe reactive moiety is exposed upon cleavage of the cleavable linker. By way of non-limiting example, the reactive group formsa covalent bond with a desired residue of the Fc region of the antibody or antigen binding fragment due to an interaction between the affinity peptide andthe Fc region. Followingthis covalentbondformation, the cleavable linker is cleaved under appropriate conditions to reveal a reactive moiety (e.g., if the cleavable linker comprises a thioester, a free sulfhydryl group is attached to the Fc region following cleavage of the cleavable linker). This new reactive moiety can then be used to sub sequently add an additional moiety, such as a conjugation handle, by way of reagent comprising the conjugation handle tethered to a sulfhydryl reactive group (e.g., alpha-halogenated carbonyl group, alpha-beta unsaturated carbonyl group, maleimide group, etc.).

[0165] In some embodiments, an affinity peptide is used to deliver a free sulfhydryl group to a lysine of the Fc region. In some embodiments, the free sulhydryl group added to the lysine of theFc region has a structure0, wherein the nitrogen atom shown is the side chain amine of the lysine. Preferably wherein there are 2 or 3 methylenes between the carbonyl andthe sulfhydryl group of the structure shown. In some embodiments, the free sulfhydryl group is then reacted with a bifunctional linking reagent to attach a new conjugation handle to the Fc region. In some embodiments, the new conjugation handle is then used to form the linker to the attached cytokine. In some embodiments, the new conjugation handle is an alkyne functional group. In some embodiments, the new conjugation handle is a DBCO functional group.

[0166] Exemplary bifunctional linking reagents useful for this purpose are of a formula A-B-C, wherein A is the sulfhydryl reactive conjugation handle (e.g., maleimide, a, P-un saturated carbonyl, a-halogenated carbonyl), B is a linkinggroup, and C is the new conjugation handle (e.g, an alkyne such as DBCO). Specific non -limiting examples of bifunctional linking reagents includeWSGR Docket No. 56146-744.601n is independently an integer from 1 -6 and each m is independently an integer from 1 -30, and related molecules (e.g., isomers). In such examples, the DBCO group is reacted with an azide group attached to the IL-18 polypeptide (e.g., at any of the residues provided herein, such asC68).

[0167] In the exemplified immunocytokines described herein (e.g., Composition A), the bifunctional linking reagent used as the structure

[0168] Alternatively, the affinity peptide can be configured such that a conjugation handle is added to the Fc region (such as by a linker group) immediately after covalent bond formation between the reactive group and a residue of the Fc region. In such cases, the affinity peptide is cleaved and the conjugation handle is immediately ready for subsequent conjugation to the IL-18 polypeptide.Alternative Methods of Modifying Fc Region

[0169] While the affinity peptide mediated modification of an Fc region of an antibody provide supra possesses many advantages over other methods which can be used to site -specifically modify the Fc region e.g., ease of use, ability to rapidly generate many different antibody conjugates, ability to use many “off-the-shelf’ commercial antibodies without the need to do timeWSGR Docket No. 56146-744.601 consuming protein engineering, etc.), other methods of performing the modification are also contemplated as being within the scope of the present disclosure.

[0170] In some embodiments, an IL-18 polypeptide can be conjugated to a suitable antibody utilizing transglutaminase-mediated site-specific antibody-drug conjugate (ADC) strategies, such as those comprising: 1) glutamine-containing tags, endogenous glutamines (e.g., native glutamines without engineering, such as glutamines in variable domains, CDRs, etc.), and / or endogenous glutamines made reactive by antibody engineering or an engineered transglutaminase; and 2) amine donor agents comprising amine donor units, linkers, and agent moieties. Nonlimiting examples of such transglutaminase mediated site-specific modifications can be found at least in publications PCT Publication No. W02020188061, US Patent Publication No. US2019194641, US Patent Publication No. US2021128743, US Patent No. US9764038, and US PatentNo. US 10434180, which are incorporatedby reference as if set forth herein in their entirety. Such strategies can be employed to either add a suitable conjugation handle to a desired site of the antibody or antigen binding fragment, or can alternatively be used to directly conjugate the IL-18 polypeptide to the antibody, though itis preferable that such a strategy is used to add a conjugation handle, then the IL-18 polypeptide is later reacted with the conjugation handle to form the immunocytokine.

[0171] In some embodiments, an IL-18 polypeptide can be conjugated to a suitable antibody utilizing a transpeptide-mediated strategy for either direct attachment of the IL-18 polypeptide to the antibody or via addition of a suitable conjugation handle to the antibody. For example, a sortase based system (such as those described in, for example, U.S. PatentNo. 10,081,684, U.S. Patent No. 10,864,277, U.S. Patent No. 11,421,022, and / or U.S. Patent No. 9,862,779) can be used, either by addition of a sortase tag to the C-terminus of antibody heavy or light chain or though a suitable linker armed with a sortase tag, to add a linker with a conjugation handle to the antibody or antigen binding fragment thereof, followed by a subsequent conjugation of the IL-18 polypeptide to the antibody or antigen binding fragment thereof using the conjugation handle.

[0172] In another aspect, the disclosure provides methods of generating immunocytokines using an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc -containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine- containing tag comprises an amino acid sequence XXQX, wherein X is any amino acid e.g., X can be the same or different amino acid), and wherein the engineered Fc-containing polypeptideWSGR Docket No. 56146-744.601 conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme).

[0173] In some embodiments, the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Ly s (e.g., the ability to form a covalent bond as an amine donor in the presence of an acyl donor and a transglutaminase) in the polypeptide or the Fc -containing polypeptide. In some embodiments, the polypeptide or the Fc-containing polypeptide comprises an amino acid modification at the last amino acid position in the carboxyl terminus relative to a wild -type polypeptide at the same position. The amino acid modification can be an amino acid deletion, insertion, substitution, mutation, or any combination thereof.

[0174] In some embodiments, the immunocytokine composition comprises a full length antibody heavy chain and an antibody light chain, wherein the acyl donor glutamine-containing tag is located at the carboxyl terminus of a heavy chain, a light chain, or both the heavy chain and the light chain.

[0175] In some embodiments, the immunocytokine composition comprises an antibody, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment. In some embodiments, the antibody is an IgG.

[0176] In another aspect, provided herein is a method for preparing an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containingtag at a carboxyl terminus, an aminoterminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX, wherein X is any amino acid (e.g., X can be the same or a different amino acid), and wherein the engineered Fc-containingpolypeptideconjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme), comprising the steps of: a) providing an engineered (Fc-containing polypeptide)-T molecule comprising the Fc- containing polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered (Fc-containing polypeptide)-T molecule in the presence of a transglutaminase; and c) allowingthe engineered (Fc-containingpolypeptide)-T to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate.

[0177] In another aspect, provided herein is a method for preparing an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine- containingtag engineered ata specific site, wherein A is an amine donor agent, wherein the amineWSGR Docket No. 56146-744.601 donor agentis site-specifically conjugated to the acyl donor glutamine-con tainingtag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence LLQGPX, wherein X is A or P (SEQ ID NO: 95), or GGLLQGPP (SEQ ID NO: 96), comprising the steps of: a) providing an engineered polypeptide-T molecule comprising the polypeptide and the acyl donor glutamine- containing tag; b) contacting the amine donor agent with the engineered polypeptide-T molecule in the presence of a transglutaminase; and c) allowing the engineered polypeptide-T to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate.

[0178] In some embodiments, the engineered polypeptide conjugate (e.g., the engineered Fc- containing polypeptide conjugate, the engineered Fab -containing polypeptide conjugate, or the engineered antibody conjugate) as described herein has conjugation efficiency of at least about 51%. In another aspect, the invention provides a pharmaceutical composition comprising the engineered polypeptide conjugate as described herein (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab -containing polypeptide conjugate, or the engineered antibody conjugate) and a pharmaceutically acceptable excipient.

[0179] In some embodiments, provided herein is a method for conjugating a moiety of interest (Z) to an antibody, comprising the steps of: (a) providing an antibody having (e.g., within the primary sequence of a constant region) at least one acceptor amino acid residue (e.g., a naturally occurring amino acid) that is reactive with a linking reagent (linker) in the presence of a coupling enzyme, e.g., a transamidase; and (b) reacting said antibody with a linking reagent (e.g., a linker comprising a primary amine) comprising a reactive group (R), optionally a protected reactive group or optionally an unprotected reactive group, in the presence of an enzyme capable of causing the formation of a covalent bond between the acceptor amino acid residue and the linking reagent (other than at the R moiety), under conditions sufficient to obtain an antibody comprising an acceptor amino acid residue linked (covalently) to a reactive group (R) via the linking reagent. Optionally, said acceptor residue of the antibody or antibody fragmentis flanked atthe +2 position by a non-aspartic acid residue. Optionally, the residue at the +2 position is a non -aspartic acid residue. In one embodiment, the residue atthe +2 position is a non-aspartic acid, non-glutamine residue. In one embodiment, the residue atthe +2 position is a non-aspartic acid, non-asparagine residue. In one embodiment, the residue atthe +2 position is a non -negatively charged amino acid (an amino acid other than an aspartic acid or a glutamic acid). Optionally, the acceptor glutamine is in an Fc domain of an antibody heavy chain, optionally further -within the CH2 domain Optionally, the antibody is free of heavy chain N297 -linked glycosylation. Optionally, theWSGR Docket No. 56146-744.601 acceptor glutamine is at position 295 and the residue at the +2 position is the residue at position 297 (EU index numbering) of an antibody heavy chain.

[0180] In one aspect, provided herein is a method for conjugating a moiety of interest (Z) to an antibody, comprisingthe steps of: (a) providing an antibody havingatleast one acceptor glutamine residue; and (b) reacting said antibody with a linker comprising a primary amine (a lysine-based linker) comprising a reactive group (R), preferably a protected reactive group, in the presence of a transglutaminase (TGase), under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked (covalently) to a reactive group (R) via said linker. Optionally, said acceptor glutamine residue of the antibody or antibody fragment is flanked at the +2 position by a non-aspartic acid residue. Optionally, the residue atthe +2 position is a non -aspartic acid residue. In one embodiment, the residue atthe +2 position is a non-aspartic acid, non-glutamine residue. In one embodiment, the residue atthe +2 position is a non-aspartic acid, non-asparagine residue. In one embodiment, the residue at the +2 position is a non -negatively charged amino acid (an amino acid other than an aspartic acid or a glutamic acid). Optionally, the acceptor glutamine is in anFc domain of an antibody heavy chain, optionally further-withinthe CH2 domain Optionally, the antibody is free of heavy chain N297-linked glycosylation. Optionally, the acceptor glutamine is at position 295 and the residue at the +2 position is the residue at position 297 (EU index numbering) of an antibody heavy chain. The antibody comprising an acceptor residue or acceptor glutamine residue linked to a reactive group (R) via a linker comprising a primary amine (a ly sine - based linker) can thereafter be reacted with a reaction partner comprising a moiety of interest (Z) to generate an antibody comprising an acceptor residue or acceptor glutamine residue linked to a moiety of interest (Z) via the linker. Thus, in one embodiment, the method further comprises a step (c): reacting (i) an antibody of step b) comprising an acceptor glutamine linked to a reactive group (R) via a linker comprising a primary amine (a lysine-based linker), optionally immobilized on a solid support, with (ii) a compound comprising a moiety of interest (Z) and a reactive group (R1) capable of reacting with reactive group R, under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked to a moiety of interest (Z) via a linker comprising a primary amine (a lysine-based linker). Preferably, said compound comprising a moiety of interest (Z) and a reactive group (R1) capable of reacting with reactive group R is provided at a less than 80 times, 40 times, 20 times, 10 times, 5 times or 4 molar equivalents to the antibody. In one embodiment, the antibody comprises two acceptor glutamines and the compound comprising a moiety of interest (Z) and a reactive group (R1) is provided at 10 or less molar equivalents to the antibody. In one embodiment, the antibody comprises two acceptor glutamines and the compound comprising a moiety of interest (Z) and a reactive group (R1) is provided at 5 or less molarWSGR Docket No. 56146-744.601 equivalents to the antibody. In one embodiment, the antibody comprises four acceptor glutamines and the compound comprising a moiety of interest (Z) and a reactive group (R1) is provided at 20 or less molar equivalents to the antibody. In one embodiment, the antibody comprises four acceptor glutamines and the compound comprising a moiety of interest (Z) and a reactive group (R1) is provided at 10 or less molar equivalents to the antibody. In one embodiment, steps (b) and / or (c) are carried out in aqueous conditions. Optionally, step (c) comprises: immobilizing a sample of an antibody comprising a functionalized acceptor glutamine residue of Formula II on a solid support to provide a sample comprising immobilized antibodies, reacting the sample comprising immobilized antibodies, optionally recovering any unreacted compound and reintroducing such recovered compound to the solid support for reaction with immobilized antibodies, and eluting the antibody conjugates to provide an antibody composition comprising a Z moiety.

[0181] In an alternative embodiment, an amino acid residue comprising a conjugation handle can be incorporated into the Fc region of the antibody (e.g., during expression of the antibody) at a desired location (e.g., any of the locations provided herein). In some embodiments, the amino acid residue comprising the conjugation handle is an unnatural amino acid.Conjugation Handle Chemistry

[0182] In some embodiments, the appropriately modified Fc region of the antibody or antigen binding fragment will comprise a conjugation handle which is used to conjugate the antibody or antigen binding fragment to an IL-18 polypeptide to produce an immunocytokine composition provided herein.

[0183] Any suitable reactive group capable of reacting with a complementary reactive group attached to the IL-18 polypeptide can be used as the conjugation handle. In some embodiments, the conjugation handle comprises a reagent for a Cu(I)-catalyzed or "copper-free" alkyne-azide triazole-forming reaction (e.g., strain promoted cycloadditions), the Staudinger ligation, inverseelectron-demand Diels-Alder (IEDDA) reaction, "photo-click" chemistry, tetrazine cycloadditions with trans-cyclooctenes, potassium acyl trifluoroborate (KAT) ligation or a metal- mediated process such as olefin metathesis and Suzuki- Miy aura or Sonogashira cross-coupling.

[0184] In some embodiments, the conjugation handle comprises a reagent for a “copper -free” alkyne azide triazole-forming reaction. Non-limiting examples of alkynes for said alkyne, azide triazole forming reaction include cyclooctyne reagents (e.g., (lR,8S,9s)-Bicyclo[6.1.0]non-4-yn- 9-ylmethanol containing reagents, dibenzocyclooctyne-amine reagents, difluorocyclooctynes, or derivatives thereof). In some embodiments, the alkyne functional group is attached to the Fc region. In some embodiments, the azide functional group is attached to the Fc region.WSGR Docket No. 56146-744.601

[0185] In some embodiments, the conjugation handle comprises a reactive group selected from azide, alkyne, tetrazine, halide, sulfhydryl, disulfide, maleimide, activated ester, alkene, aldehyde, ketone, imine, hydrazine, potassium acyl trifluoroborate, hydroxylamine (e.g., O-substituted hydroxylamine) and hydrazide. In some embodiments, the IL-18 polypeptide comprises a reactive group complementary to the conjugation handle of the Fc region. In some embodiments, the conjugation handle and the complementary conjugation handle comprise “CLICK” chemistry reagents. Exemplary groups of click chemistry residue are shown in Hein etal. , “Click Chemistry, A Powerful Tool for Pharmaceutical Sciences,” Pharmaceutical Research, volume 25, pages 2216-2230 (2008); Thirumurugan etal., “Click Chemistry for Drug Development and Diverse Chemical-Biology Applications,” Chem. Rev. 2013, 113, 7, 4905-4979; US20160107999A1; US10266502B2; and US20190204330A1, each of which is incorporated by reference in its entirety.Linker Structure

[0186] In some embodiments, the linker used to attach the antibody or antigen binding fragment and the IL-18 polypeptide comprises points of attachment at both moieties. The points of attachment can be any of the residues for facilitatingthe attachment as provid edherein. The linker structure can be any suitable structureforcreatingthe spatial attachmentbetweenthe two moieties. In some embodiments, the linker provides covalent attachment of both moieties. In some embodiments, the linker is a chemical linker (e.g., not an expressed polypeptide as in a fusion protein). In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-peptide linker (e.g., does not consist of amino acid residues).Chemical Linkers

[0187] In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker comprises at least one portion which is not comprised of amino acid residues. In some embodiments, the linker comprises a polymer. In some emb odiments, the linke r comprises a water soluble polymer. In some embodiments, the linker comprises poly(alkylene oxide), polysaccharide, poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof. In some embodiments, the linker comprises poly(alkylene oxide). In some embodiments, the poly(alkylene oxide) is polyethylene glycol or polypropylene glycol, or a combination thereof. In some embodiments, the poly(alkylene oxide) is polyethylene glycol.

[0188] In some embodiments, the linker is a bifunctional linker. In some embodiments, the bifunctional linker comprises an amide group, an ester group, an ether group, a thioether group, or a carbonyl group. In some embodiments, the linker comprises a non -polymer linker. In someWSGR Docket No. 56146-744.601 embodiments, the linker comprises a non -polymer, bifunctional linker. In some embodiments, the non-polymer, bifunctional linker comprises succinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate; Maleimidocaproyl; Valine-citrulline; Allyl(4-methoxyphenyl)dimethylsilane; 6- (Allyloxycarbonylamino)-l-hexanol; 4-Aminobutyraldehyde diethyl acetal; or (E)-N-(2- Aminoethyl)-4-{2-[4-(3 -azidopropoxy )phenyl]diazenyl}benzamide hydrochloride.

[0189] The linker can be branched or linear. In some embodiments, the linker is linear. In some embodiments, the linker is branched. In some embodiments, the linker comprises a linear portion (e.g., between the first point of attachment and the second point of attachment) of a chain of at least 10, 20, 50, 100, 500, 1000, 2000, 3000, or 5000 atoms. In some embodiments, the linker comprises a linear portion of a chain of at least 10, 20, 30, 40, or 50 atoms. In some embodiments, the linker comprises a linear portion of at least 10 atoms. In some embodiments, the linker is branched and comprises a linear portion of a chain of at least 10, 20, 50, 100, 500, 1000, 2000, 3000, or 5000 atoms. In some embodiments, the linker comprises a linear portion of at from 1 to 1000 atoms, 1 to 900 atoms, 1 to 800 atoms, 1 to 500 atoms, 1 to 400 atoms, 1 to 300 atoms, 1 to 200 atoms, 1 to 100 atoms, 1 to 50 atoms, 10 to 1000 atoms, 10 to 900 atoms, 10 to 800 atoms, 10 to 500 atoms, 10 to 400 atoms, lOto 300 atoms, lOto 200 atoms, 10 to 100 atoms, 10 to 50 atoms, 25 to 1000 atoms, 25 to 900 atoms, 25 to 800 atoms, 25 to 500 atoms, 25 to 400 atoms, 25 to 300 atoms, 25 to 200 atoms, 25 to 100 atoms, 25 to 50 atoms, 50 to 1000 atoms, 50 to 900 atoms, 50 to 800 atoms, 50 to 500 atoms, 50 to 400 atoms, 50 to 300 atoms, 50 to 200 atoms, or 50 to 100 atoms. In some embodiments, the linker has a linear length of from about 10 angstroms to about 200 angstroms. In some embodiments, the linker has a linear length of from about 10 to 500, 10 to 200, 10 to 150, 10 to 125, 10 to 100, 10 to 75, 10 to 50, 25 to 200, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 150, 50 to 100, or 50 to 75 angstroms.

[0190] In some embodiments, the linker has a molecular weight of about 200 Daltons to about 2000 Daltons. In some embodiments, the linker has a molecular weight of about 200 Daltons to about 5000 Daltons. In some embodiments, the linker has a molecular weight of 200 Daltons to 100,000 Daltons. In some embodiments, the linker has a molecular weight of at least about 500 Daltons, atleast about 1,000 Daltons, at least about 5,000 Daltons, atleast about 10,000 Daltons, at least about 15,000 Daltons, atleast about 20,000 Daltons, atleast about 25,000 Daltons, or at least about 30,000 Daltons. In some embodiments, the linker as a molecular weight of at most about 100, OOODaltons, at most about 50, 000 Daltons, atmostabout40,000Daltons, atmostabout 30,000 Daltons, at most about 25,000 Daltons, atmost about 20,000 Daltons atmost about 15,000 Daltons, at most about 10,000 Daltons, or at most about 5,000 Daltons.WSGR Docket No. 56146-744.601

[0191] In some embodiments, the linker comprises a reaction product of one or more pairs of conjugation handles and a complementary conjugation handle thereof. In some embodiments, the reaction product comprises a triazole, a hydrazone, pyridazine, a sulfide, a disulfide, an amide, an ester, an ether, an oxime, an alkene, or any combination thereof. In some embodiments, the reaction product comprises a triazole. The reaction product can be separated from the first point of attachment and the second point of attachment by any portion of the linker. In some embodiments, the reaction product is substantially in the center of the linker. In some embodiments, the reaction product is substantially closer to one point of attachment than the other is.

[0192] In some embodiments, the linker comprises a structure of Formula (X)wherein each of L1, L2, L3, L4, L5, L6, L7L8, and L9is independently -O-, -NRL-, -(Ci-Ce alkylene)NRL-, -NRL(C!-C6alkylene)-, -N(RL)2+-,-(C C6alkylene)N(RL)2+-, -N(RL)2+-(CrC6alkylene)-, -OP(=O)(ORL)O-, -S-, -(CrC6alkylene)S-, -S(CrC6alkylene)-, -S(=O)-, - S(=O)2-, -C(=O)-, -(Ci-C6alkylene)C(=O)-, -C(=O) (Ci-C6alkylene)-, -C(=O)O-, -OC(=O)- -OC(=O)O-, -C(=O)NRL-, -C(=O)NRL(CI-C6alkylene)-, -(Ci-C6alkylene)C(=O)NRL-, - NRLC(=O)-, -(Ci-Cg alkylene)NRLC(=O)-, -NRLC(=O)(C C6alkylene)-, -OC(=O)NRL-, - NRLC(=O)O-, -NRLC(=O)NRL-, -NRLC(=S)NRL-, -CRL=N-, -N=CRL, -NRLS(=O)2-, - S(=O)2NRL-, -C(=O)NRLS(=O)2-, -S(=O)2NRLC(=O)-, substituted or unsubstituted Ci-Ce alkylene, substituted or un substituted Ci-C6heteroalkylene, substituted or un substituted C2- C& alkenylene, substituted or un substituted C2-Ce alkynylene, substituted or unsubstituted C&- C2o arylene, substituted or unsubstituted C2-C2o heteroarylene, -(CH2-CH2-O)qa-, -(O-CH2- CH2)qb-, -(CH2-CH(CH3)-O)qc-, -(O- CH(CH3)-CH2)qd-, a reaction product of a conjugation handle and a complementary conjugation handle, or absent; each RLis independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C2-C5alkynyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C2-C7heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and each of qa, qb, qc and qd is independently an integer from 1 -100, wherein each ' is a point of attachment to the antibody or antigen binding fragment or the IL- 18 polypeptide.WSGR Docket No. 56146-744.601

[0193] In some embodiments, the linker consists of a plurality of structures of Formula (X) to form the linkage between the antibody or antigen binding fragment and the IL-18 polypeptide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more structures of Formula (X) appendedfrom end to end,where only the terminal denote points of attachment to the antibody or antigen binding fragment or the IL-18 polypeptide).

[0194] In some embodiments, the polymer comprises a linker comprising a structure of Formula(X’)wherein each L’ is independently -O-, -NRL-, -(Ci-C6alkylene)NRL-, -NRL(CI-C6alkylene)-, - N(RL)2+-, -(C C6alkylene)N(RL)2+-, -N(RL)2+-(CrC6alkylene)-, -OP(=O)(ORL)O-, -S-, - (Ci-C6alkylene)S-, -S(Ci-C6alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(Ci-C6alkylene)C(=O)- , -C(=O) (CrC6alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NRL-, -C(=O)NRL(C!- C6alkylene)-, -(Ci-C6alkylene)C(=O)NRL-, -NRLC(=O)-, -(Ci-C6alkylene)NRLC(=O)-, - NRLC(=O)(C1-C6alkylene)-, -OC(=O)NRL-, -NRLC(=O)O-, -NRLC(=O)NRL-, - NRLC(=S)NRL-, -CRL=N-, -N=CRL, -NRLS(=O)2-, -S(=O)2NRL-, -C(=O)NRLS(=O)2-, - S(=O)2NRLC(=O)-, substituted or unsubstituted Ci-C6alkylene, substituted or unsubstituted Ci-Ce heteroalkylene, substituted or unsubstituted C2-Ce alkenylene, substituted or unsubstituted C2-C6alkynylene, substituted or unsubstituted C6-C20arylene, substituted or unsubstituted C2-C20heteroarylene, -(CH2-CH2-O)qa-, -(O-CH2-CH2)qb-, -(CH2-CH(CH3)- O)qc-, -(O- CH(CH3)-CH2)qd-, a reaction product of a conjugation handle and a complementary conjugation handle, or absent; (Ci-C6alkylene); each RLis independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-Ce alkenyl, substituted or unsubstituted C2-C5alkynyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted C2-C7heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and each of qa, qb, qc and qd is independently an integer from 1 -100, g is an integer from 1 -100,wherein each is a point of attachment to the IL-18 polypeptide or the antibody or antigen binding fragment.WSGR Docket No. 56146-744.601

[0195] In some embodiments, the linker of Formula (X) or Formula (X’) comprises the structure:° is the attachment to a lysine residue of the antibody or antigen binding fragment;L is a linking group; andpoint of attachment to a linking group which connects to point of attachment on the IL- 18 polypeptide, or a regioisomer thereof.

[0196] In some embodiments, L has a structureWSGR Docket No. 56146-744.601wherein each n is independently an integer from 1 -6 and each m is an integer from 1 -30. In some embodiments, each m is independently 2 or 3. In some embodiments, each m is an integer from 1-24, from 1-18, from 1-12, or from 1-6.

[0197] In some embodiments, the linker of Formula (X) or of Formula (X’) comprises the structure:° is the first point of attachment to a lysine residue of the polypeptide which selectively binds to PD-1;L” is a linking group; andpoint of attachment to a linking group which connects to the first point of attachment, or a regioisomer thereof.

[0198] In some embodiments, L” has a structureWSGR Docket No. 56146-744.601independently an integer from 1 -6 and each m is independently an integer from 1 -30. In some embodiments, each m is independently 2 or 3. In some embodiments, each m is an integer from 1-24, from 1-18, from 1-12, or from 1-6.

[0199] In some embodiments, L or L” comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more subunits each independently, wherein each n is independently an integer from 1 -30. In some embodiments, each n is independently an integer from 1 -6. In some embodiments, L or L” comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the subunits.

[0200] In some embodiments, L or L” is a structure of Formula (X”)wherein each of Lla, L2a, L3a, L4a, L5a, is independently -O-, -NRLa-, -(Ci-Ce alkylene)NRLa-, - NR^Ci-Cg alkylene)-, -N(RL)2+-, -(C C6alkylene)N(RLa)2+(C1-C6alkylene)-, -N(RL)2+-, - OP(=O)(ORLa)O-, -S-, -(Ci-C6alkylene)S-, -S(Ci-C6alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6alkylene)C(=O)-, -C(=O)(Ci-C6alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, - C(=O)NRLa-, -C(=O)NRLa(C1-C6alkylene)-, -(C C6alkylene)C(=O)NRLa-, -NRLaC(=O)-, - (C C6alkylene)NRLaC(=O)-, -NRLaC(=O)(C1-C6alkylene)-, -OC(=O)NRLa-, -NRLaC(=O)O- , -NRLaC(=O)NRLa-, -NRLaC(=S)NRLa-, -CRLa=N-, -N=CRLa, -NRLaS(=O)2-, -S(=O)2NRLa-, - C(=O)NRLaS(=O)2-, -S(=O)2NRLaC(=O)-, substituted or unsubstituted Ci-C6alkylene,WSGR Docket No. 56146-744.601 substituted or unsubstituted Ci-C6heteroalkylene, substituted or unsubstituted C2-C6alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C20 arylene, substituted or un substituted C2-C2o heteroarylene, -(CH2-CH2-O)qe-, -(O-CH2-CH2)qf- , -(CH2-CH(CH3)-O)qg-, -(O- CH(CH3)-CH2)qh-, a reaction product of a conjugation handle and a complementary conjugation handle, or absent; (Ci-Ce alkylene) each RLais independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-Cg cycloalkyl, substituted or unsubstituted C2-C7heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and each of qe, qf, qg and qh is independently an integer from 1 -100.

[0201] In some embodiments, L or L” comprises a linear chain of 2 to 10, 2 to 15, 2 to 20, 2 to 25, or 2 to 30 atoms. In some embodiments, the linear chain comprises one or more alkyl groups (e.g., lower alkyl (C1-C4)), one or more aromatic groups (e.g., phenyl), one or more amide groups, one or more ether groups, one or more ester groups, or any combination thereof.

[0202] In some embodiments, the linking group which connects to the first point of attachment (e.g., the point of attachment to the IL-18 polypeptide) comprises poly(ethylene glycol). In some embodiments, the linking group comprises about 2 to about 30 poly (ethylene glycol) units. In some embodiments, the linking group which connects to the first point of attachment (e.g., the point of attachment to the IL- 18 polypeptide) is a functionality attached to a cytokine provided herein which comprises an azide (e.g., the triazole is the reaction product of the azide).

[0203] In some embodiments, each reaction product of a conjugation handle and a complementary conjugation handle independently comprises a triazole, a hydrazone, pyridazine, a sulfide, a disulfide, an amide, an ester, an ether, an oxime, or an alkene . In some embodiments, each reaction product of a conjugation handle and a complementary conjugation handle comprises a triazole. In some embodiments, each reaction product of a conjugation handle and a complementary conjugation handle comprise a structureWSGR Docket No. 56146-744.601or a regioisomer or derivative thereof.the carbonyl on the left side of the molecule is attached to an Fc domain lysine (e.g., K248) of the anti-PD-1 antibody or antigen binding fragment thereof and the right side of the molecule is attached to a cysteine of the IL-18 polypeptide (e.g., C68). In exemplified versions of the immunocytokines provided herein, the linker has the structurePeptide Linkers

[0205] In some embodiments, the antibody or antigen binding fragment is linked to the IL-18 polypeptide through a peptide linker. In some embodiments, the antibody or antigen binding fragment is linked to the IL-18 polypeptide as a fusion protein. In such instances, the linker comprises one or more peptide bonds between the antibody or antigen binding fragment and the IL- 18 polypeptide. In some embodiments, the linker between the fusion protein of the antibody or antigen binding fragment and the IL-18 polypeptide is a bond. In some embodiments, the linker between the fusion protein of the antibody or antigen binding fragment and the IL-18 polypeptide is a linking peptide. Non-limiting examples of linking peptides include, but are not limited to (GS)n(SEQ ID NO: 124), (GGS)n(SEQ ID NO: 125), (GGGS)n(SEQ ID NO: 126), (GGSG)nWSGR Docket No. 56146-744.601(SEQ ID NO: 127), or (GGSGG)n(SEQ ID NO: 128), (GGGGS)n(SEQ ID NO: 129), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, a linking peptide can be (GGGGS)s (SEQ ID NO: 130) or (GGGGS)4(SEQ ID NO: 123). In some embodiments, the IL-18 polypeptide is fused to the C-terminal end of the antibody or antigen binding fragment (optionally through a linking peptide). In some embodiments, the IL- 18 polypeptide is fused to the N-terminal end of the antibody or antigen binding fragment (optionally through a linking peptide).Cleavable linkers

[0206] In some embodiments, the linker (e.g., a chemical or peptide linker as provided herein) is a cleavable linker. In some embodiments, the cleavable linker is cleaved at, near, or in a tumor microenvironment. In some embodiments, the tumor is mechanically or physically cleaved at, near, or in the tumor microenvironment. In some embodiments, the tumor is chemically cleaved at, near, or in a tumor microenvironment. In some embodiments, the cleavable linker is a reduction sensitive linker. In some embodiments, the cleavable linker is an oxidation sensitive linker. In some embodiments, the cleavable linker is cleaved as a result of pH at, near, or in the tumor microenvironment. In some embodiments, the cleavable linker is cleaved by a tumor metabolite at, near, or in the tumor microenvironment. In some embodiments, the cleavable linker is cleaved by a protease at, near, or in the tumor microenvironment. However, in preferred embodiments of the instant disclosure, the linker is not one which is cleaved in target tissue (e.g., the linker is not designed to release the IL-18 polypeptide from the anti-PD-1 antibody or antigen binding fragment thereof).IL-18 Polypeptides

[0207] The present disclosure describes antibodies or antigen binding fragments linked to interleukin- 18 (IL-18) polypeptides as immunocytokine compositions and their use as human therapeutic agents. IL-18 is a pro-inflammatory cytokine that elicits biological activities that initiate or promote host defense and inflammation following infection or injury. IL- 18 has been implicated in autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, hemophagocytic syndromes, macrophage activation syndrome, sepsis, and acute kidney injury. In some models of disease, IL-18 plays a protective role.

[0208] IL-18 also plays a major role in the production of IFNy from T-cells and natural killer cells. IFNy is a T helper type 1 cytokine mainly produced by T cells, NK cells, and macrophages and is critical for innate and adaptive immunity against viral, some bacterial, and protozoal infections. IFNy is also an important activator of macrophages and inducer of Class II major histocompatibility complex (MHC) molecule expression.WSGR Docket No. 56146-744.601

[0209] IL-18 forms a signaling complex by binding to the IL-18 alpha chain (IL-18Ra), which is the ligand binding chain for mature IL-18. However, the binding affinity of IL-18 to IL-18Ra is low. In cells that express the co-receptor, IL-18 receptor beta chain (IL-18RP), a high affinity heterodimer complex is formed, which then activates cell signaling.

[0210] The activity of IL-18 is balanced by the presence of a high affinity, naturally occurring IL- 18 binding protein (IL-18BP). IL-18BP binds IL-18 and neutralizes the biological activity of IL- 18. Cell surface IL-18Ra competes with IL-18BP for IL-18 binding. Increased disease severity can be associated with an imbalanceof IL-18 to IL-18BP such that levels of free IL-18 are elevated in the circulation. FIGURE 4 illustrates the mechanism of action of IL-18, IFNy production, IL- 18BP production, and inhibition of IL-18 activity by IL-18BP. IL-18 induces IFNy production, which in turn induces IL- 18BP production. IL-18BP then competes with IL-18Ra to inhibit IL-18 activity.

[0211] In some embodiments, the IL-18 polypeptides of the immunocytokines provided herein display reduced binding to IL-18BP and retain binding to the IL-18 receptor. The IL-18 polypeptides with this property provided herein are able to retain IL-18 receptor signaling activity (including inducing production of IFNy) even in the presence of IL-18BP. This allows the immunocytokines provided herein to retain IL-18 signaling activity well beyond a short period of time after administration, or upon repeat administrations. In some embodiments, the IL-18 polypeptides with this property comprise a modification (e.g., substitution, polymer attachment, or deletion) at one or more amino acid residues which convey this property to the IL-18 polypeptide. Examples of IL-18 polypeptides with this property are provided herein, as well as those otherwise known, such as those described in Patent Cooperation Treaty Publication No. W02019051015A1, which is hereby incorporated by reference as if set forth herein in its entirety. In addition to IL-18 polypeptides provided herein, these otherwise known IL- 18 polypeptides or their analogs may similarly be modified with points of attachmentto the linker as provided herein. In some embodiments, an IL-18 can by prepared synthetically, such as according to the route shown in FIGURE 5.Points of Attachment of Chemical Linkers to IL-18 Polypeptides

[0212] The immunocytokines provided herein comprise linkers which have a point of attachment to the IL-18 polypeptide. In some embodiments, the linker is a chemical linker. As discussed supra, the linker has another point of attachmentto the antibody or antigen binding fragment at any residue as provided herein. The point of attachmentto the IL-18 polypeptide is to a residue as provided herein.WSGR Docket No. 56146-744.601

[0213] In some embodiments, the linker is attached to an amino acid residue of the IL-18 polypeptide. In some embodiments, the linker is attached to any amino acid residue of the IL-18 polypeptide (e.g., at a position corresponding to any one of positions 1 -157 of SEQ ID NO: 1). In some embodiments, the linker is attached at a non-terminal residue of the IL-18 polypeptide (e.g, a residue at position corresponding to any one of positions 2-156 of SEQ ID NO: 1). In some embodiments, the linker is attached at a non-terminal residue of the IL- 18 polypeptide, wherein the IL- 18 polypeptide has been extended or truncated by one or more amino acids relative to SEQ ID NO: 1.

[0214] In some embodiments, the linker is attached to the IL-18 polypeptide at a residue in a region comprising residues 2-156, wherein residue position numbering is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue in a region comprising residues 30-150. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue in a region comprising residues 33 -43, residues 60-100, residues 65-75, residues 80-90, residues 85-100, residues 90-110, residues 115-130, residues 120- 130, or residues 140-150. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue selected from residue 38, 68, 69, 70, 76, 78, 85, 86, 95, 98, 121, 127, and 144. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue selected from 68, 69, 70,85, 86, and 98. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 68, 69, or 70. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 85,86, 95, or 98. In some embodiments, the linkeris attached to the IL-18 polypeptide at residue 68. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 69. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 70. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 85. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 86. In some embodiments, the linkeris attached to the IL-18 polypeptide at residue 95. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 98.

[0215] In some embodiments, the linker is attached to the IL-18 polypeptide at a residue which is known in the art to be compatible with attachment of a polymer to the IL-18 polypeptide without having a profound impact on the bioactivity of the IL-18 polypeptide. Examples of these residues include residues 38, 76, 78, 121, 127, and 144, as describedin PCT Pub. No. W02004091517A2, which is hereby incorporated by reference as if set forth in its entirety.

[0216] In some embodiments, the residue to which the linker is attached is a natural amino acid residue. In some embodiments, the residue to which the linker is covalently attached is selected from cysteine, aspartate, asparagine, glutamate, glutamine, serine, threonine, lysine, and tyrosine.WSGR Docket No. 56146-744.601In some embodiments, the residue to which the linker is covalently attached is selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, lysine, and tyrosine. In some embodiments, the linker is covalently attached to a cysteine. In some embodiments, the linker is covalently attached to a lysine. In some embodiments, the linker is covalently attached to a glutamine. In some embodiments, the linker is covalently attached to an asparagine. In some embodiments, the residue to which the linker is attached is a tyrosine. In some embodiments, the residue to which the linker is attached is the natural amino acid in that position in SEQ ID NO: 1.

[0217] In some embodiments, the linker is attached to a different natural amino acid which is substituted at the relevant position. The substitution can be for a naturally occurring amino acid which is more amenable to attachment of additional functional groups (e.g., aspartic acid, cysteine, glutamic acid, lysine, serine, threonine, or tyrosine), a derivative of modified version of any naturally occurring amino acid, or any unnatural amino acid (e.g., an amino acid containing a desired conjugation handle, such as a CLICK chemistry reagent such as an azide, alkyne, etc. . In some embodiments, the linker is covalently attached to site -specifically to a natural amino acid.

[0218] In some embodiments, the linker is attached at an unnatural amino acid residue. In some embodiments, the unnatural amino acid residue comprises a conjugation handle. In some embodiments, the conjugation handlefacilitatesthe addition of the linker to the IL-18 polypeptide. The conjugation handle can be any of the conjugation handles provided herein. In some embodiments, the linker is covalently attached site -specifically to the unnatural amino acid. Nonlimiting examples of amino acid residues comprising conjugation handles can be found, for example, in PCT Pub. Nos. WO2015054658A1, WO2014036492 Al, WO2021133839A1 W02006069246 A2, and W02007079130A2, each of which is incorporated by reference as if set forth in its entirety.

[0219] In some embodiments, the linker is covalently attached at residue 68. In some embodiments, the linker is covalently attached at residue C68, C68E, C68D, C68Q, C68K, C68N, or C68Y. In some embodiments, the linker is covalently attached at residue C68. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 68. In preferred embodiments ofthe instant disclosure, the linker is attached to residue C68 oftheIL-18 polypeptide (e.g., C68 of SEQ ID NO: 30, for example).

[0220] In some embodiments, the linker is covalently attached at residue 69. In some embodiments, the linker is covalently attached at residue E69, E69C, E69D, E69Q, E69K, E69N, or E69Y. In some embodiments, the linker is covalently attached at residue E69. In some embodiments, the linker is covalently attached residue E69C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 69.WSGR Docket No. 56146-744.601

[0221] In some embodiments, the linker is covalently attached at residue 70. In some embodiments, the linker is covalently attached at re sidueK70, K70C,K70D, K70Q, K70E, K70N, or K70Y. In some embodiments, the linker is covalently attached at residue K70. In some embodiments, the linker is covalently attached residue K70C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 70.

[0222] In some embodiments, the linker is covalently attached at residue 85. In some embodiments, the linker is covalently attached at residue E85, E85C, E85D, E85Q, E85K, E85N, or E85Y. In some embodiments, the linker is covalently attached at residue E85. In some embodiments, the linker is covalently attached residue E85C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 85.

[0223] In some embodiments, the linker is covalently attached at residue 86. In some embodiments, the linker is covalently attached at residue M86C, M86D, M86Q, M86K, M86N, M86E, or M86Y. In some embodiments, the linker is covalently attached M86C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 86.

[0224] In some embodiments, the polymer is covalently attached at residue 95. In some embodiments, the polymer is covalently attached at residue T95, T95C, T95D, T95Q, T95K, T95N, T95E, or T95 Y. In some embodiments, the polymer is covalently attached at residue T95C, T95D, T95Q, T95K, T95N, T95E, or T95Y. In some embodiments, the polymer is covalently attached at residue T95C. In some embodiments, the polymer is covalently attached to an unnatural amino acid at residue 95.

[0225] In some embodiments, the linker is covalently attached at residue 98. In some embodiments, the linker is covalently attached at residue D98, D98C,D98Q, D98K, D98N,D98E, or D98Y. In some embodiments, the linker is covalently attached at residue D98C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 98.

[0226] In some embodiments, the linker is covalently attached through a modified natural amino acid. In some embodiments, the modified natural amino acid comprises a conjugation handle. In some embodiments, the linker is covalently attached through a modified amino acid a. In some embodiments, the modified amino acid a is an amino-acid-PEG-azide group. In some embodiments, the modified amino acid a is a glutamate, aspartate, lysine, cysteine, or tyrosine modified to incorporate an azide group linked to the amino acid through a PEG spacer. In some embodiments, the modified amino acid a has a structure selected from:WSGR Docket No. 56146-744.601wherein each n is independently an integer from 1 -30. In some embodiments, n is an integer from 1-20, 1-10, 2-30, 2-20, 2-10, 5-30, 5-20, or 5-10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 12. The modified amino acid a can be incorporated at any point of attachment of the IL-18 polypeptide as provided herein. In some embodiments, the modified amino acid a is located at a position on the IL-18 polypeptide selected from residue 68, residue 69, residue 70, residue 85, residue 86, residue 95, or residue 98.

[0227] Where IL-18 polypeptides contain unnatural amino acids or modified natural amino acids (e.g., those provided herein for purposes of conjugation), these amino acids may be incorporated into the IL-18 polypeptides using many techniques known in the art for introduction such modifications. For example, recombinant proteins with unnatural amino acids can be made using methods as described in Patent Cooperation Treaty Publication Nos. WO2016115168, W02002085923, W02005019415, and W02005003294. Alternatively or in combination, unnatural or modified natural amino acids can be incorporated into chemically synthesized proteins during synthesis.Modifications to IL-18 polypeptides

[0228] In some embodiments, the IL-18 polypeptide of the immunocytokine comprises one or more modifications to that of SEQ ID NO: 1. The modifications provided herein are in addition to any modification at the point of attachment as discussed supra. In some embodiments, the residue position numbering of the IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence.WSGR Docket No. 56146-744.601

[0229] Modifications to the IL-18 polypeptide described herein encompass mutations, addition of various functionalities, deletion of amino acids, addition of amino acids, or any other alteration of the wild-type version of the protein or protein fragment. Functionalities which may be added to polypeptides include polymers, linkers, alkyl groups, detectable molecules such as chromophores or fluorophores, reactive functional groups, or any combination thereof. In some embodiments, functionalities are added to individual amino acids of the polypeptides. In some embodiments, functionalities are added site-specifically to the polypeptides.

[0230] In some embodiments, the IL-18 polypeptide of the immunocytokine comprise one or more modifications in addition to a modification neededto attach the linker to the relevant residue of the IL-18 polypeptide (e.g., an amino acid substitution at a residue to which the linker is not attached). In some embodiments, the modification is in the range of amino acid residues 1 -127, based on the sequence of human IL- 1837-193(SEQ ID NO: 1). SEQ ID NO: 1 reflects the bioactive form of IL-18. Endogenously, IL-18 is initially expressed with an additional 36 amino acid segment at the N-terminus which is cleaved by caspases to mediate biologic activity.

[0231] In some embodiments, the IL-18 polypeptide of the immunocytokine described herein contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more modified amino acid residues.

[0232] In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1.

[0233] In some embodiments, the IL- 18 polypeptide of the immunocytokine provided herein comprises an amino acid sequence of any one of SEQ ID NOs: 2-72 provided herein. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of any one of SEQ ID NOs: 2-72. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 30. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence of SEQ ID NO: 59. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 59.

[0234] In some embodiments, the IL-18 polypeptide of the immunocytokine described herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 amino acid substitutions, wherein the amino acid substitutions are relative to SEQ ID NO: 1. In some embodiments, the IL- 18 polypeptide comprises 1 to 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 1 or 2 amino acid substitutions, 1 to 3 amino acidWSGR Docket No. 56146-744.601 substitutions, 1 to 4 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 6 amino acid substitutions, 1 to 7 amino acid substitutions, 1 to 8 amino acid substitutions, 2 to 3 amino acid substitutions, 2 to 4 amino acid substitutions, 2 to 5 amino acid substitutions, 2 to 6 amino acid substitutions, 2 to 7 amino acid substitutions, 2 to 8 amino acid substitutions, 2 to 9 amino acid substitutions 3 or 4 amino acid substitutions, 3 to 5 amino acid substitutions, 3 to 6 amino acid substitutions, 3 to 7 amino acid substitutions, 3 to 9 amino acid substitutions, 4 or 5 amino acid substitutions, 4 to 6 amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 amino acid substitutions, 5 or 6 amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 amino acid substitutions, 6 or 7 amino acid substitutions, 6 to 9 amino acid substitutions, or 7 to 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises at most 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions.

[0235] In some embodiments, one modification is at amino acid residue 6. In some embodiments, one modification is in the range of amino acid residues 53 -63. In some embodiments, one modification is at amino acid residue 53. In some embodiments, one modification is at amino acid residue 63.

[0236] In some embodiments, the IL- 18 polypeptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 1 selected from: Y0 IX, F02X, E06X, S10X, VI IX, D17X, C38X, M51X, K53X, D54X, S55X, T63X, C68X, C76X, AND C127X, wherein each X is independently a natural or non-natural amino acid. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at the point of attachment of the linker, such as residue 69, residue 70, residue 85, residue 86, residue 95, orresidue 98. In some embodiments, the IL-18 polypeptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 1 selected from: Y01G, F02A, E06K, S10T, VI II, D17N, C38S, C38A, C38Q, M51G, K53A, D54A, S55A, T63A, C68S, C68A, C76S, C76A, C127A, and C127S. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at the point of attachment of the linker, such as E69C, K70C, E85C, M86C, T95C, or D98C.

[0237] In some embodiments, the interleukin- 18 (IL-18) polypeptide comprising E06K and K53A, wherein residue position numbering of the IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the IL-18 polypeptide further comprises VI II. In some embodiments, the IL-18 polypeptide further comprises T63 A. In some embodiments, the IL-18 polypeptide further comprises at least one of Y01X, S55X, F02X, D54X, C38X, C68X,WSGR Docket No. 56146-744.601E69X, K70X, C76X, or Cl 27 X, wherein each X is independently an amino acid or an amino acid derivative. In some embodiments, the IL-18 polypeptide further comprises at least one of Y01G, S55A, F02A, D54A, C38S, C38A, C38Q, C68S, C68A, E69C, K70C, C76S, C76A, C127S, or Cl 27 A. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at the point of attachment of the linker, such as residue 69, residue 70, residue 85, residue 86, residue 95, or residue 98.

[0238] In some embodiments, the IL-18 peptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 1, wherein the modification is E06X, VI IX, K53X, S55X, orT63X, wherein X is a natural or non-natural amino acid. In some embodiments, the IL-18 peptide comprises at least two modifications to the amino acid sequence of SEQ ID NO: 1 , wherein the modifications comprise E06X andK53X; E06X and S55X; K53X and S55X; E06X and T63X; or K53X and T63X, wherein X is a natural or non-natural amino acid. In some embodiments, the IL- 18 peptide comprises at least three modifications to the amino acid sequence of SEQ ID NO: 1, wherein the modifications comprise E06X, K53X, and S55X; orE06X, K53X, andT63X, wherein X is a natural or non-natural amino acid. In some embodiments, the IL-18 peptide comprises at least four modifications to the amino acid sequence of SEQ ID NO: 1 , wherein the modifications comprise E06X, K53X, S55X, and T63X; E06X, K53X, S55X, andYOIX; E06X, K53X, S55X, and F02X; E06X, K53X, S55X, and D54X; E06X, K53X, S55X, and M51X; or C38X, C68X, C76X, and C127X, wherein Xis a natural or non -natural amino acid. In each embodiment wherein a plurality of amino acids residues are replaced with a natural or non-natural amino acid X, each X is independently the same or a different amino acid.

[0239] In certain embodiments, the IL-18 polypeptide comprises a substitution at residue Yl. In certain embodiments, the IL-18 polypeptide can comprise YIM substitution. Unless specifically mentioned otherwise, the residue position numbering is provided in this paragraph, and elsewhere in this disclosure is based on SEQ ID NO: 1, as a reference sequence. Unless specifically mentioned otherwise, the amino acid substitutions provided in this paragraph, and elsewhere in this disclosure is with respect to SEQ ID NO: 1 , as a reference sequence. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue F2. In certain embodiments, the IL-18 polypeptide can comprise F2A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue E6. In certain embodiments, the IL-18 polypeptide comprises E6K substitution. In certain embodiments, the IL-18 polypeptide comprises E6R substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue K8. In certain embodiments, the IL-18 polypeptide comprises K8L substitution. In certain embodiments, the IL- 18 polypeptide comprises K8E substitution. In certain embodiments, the IL-18 polypeptideWSGR Docket No. 56146-744.601 comprises K8R substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue VI 1. In certain embodiments, the IL-18 polypeptide can comprise VI II substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue E31. In certain embodiments, the IL-18 polypeptide comprises E31 A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue T34. In certain embodiments, the IL-18 polypeptide comprises T34A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue D35. In certain embodiments, the IL-18 polypeptide comprises D35A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue S36. In certain embodiments, the IL- 18 polypeptide comprises S36A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue D37. In certain embodiments, the IL-18 polypeptide comprises D37A substitution. In certain embodiments, the IL- 18 polypeptide comprises a substitution at residue D40. In certain embodiments, the IL-18 polypeptide comprisesD40A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue N41. In certain embodiments, the IL-18 polypeptide comprises N41A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue 149. In certain embodiments, the IL-18 polypeptide comprises 149E substitution. In certain embodiments, the IL-18 polypeptide comprises I49M substitution. In certain embodiments, the IL-18 polypeptide comprises I49R substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residueK53. In certain embodiments, the IL-18 polypeptide comprises K53A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution atresidueD54. In certain embodiments, the IL-18 polypeptide comprises D54A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue S55. In certain embodiments, the IL-18 polypeptide comprises S55A substitution. In certain embodiments, the IL-18 polypeptide comprises S55T substitution. In certain embodiments, the IL-18 polypeptide comprises S55H substitution. In certain embodiments, the IL-18 polypeptide comprises S55R substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue T63. In certain embodiments, the IL-18 polypeptide comprises T63A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue Q103. In certain embodiments, the IL-18 polypeptide comprises QI 03R substitution. In certain embodiments, the IL- 18 polypeptide comprises Q103E substitution. In certain embodiments, the IL-18 polypeptide comprises QI 03K substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue G108. In certain embodiments, the IL-18 polypeptide comprises G108A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue H109. In certain embodiments, the IL-18 polypeptideWSGR Docket No. 56146-744.601 comprises Hl 09 A substitution. In certain embodiments, the IL- 18 polypeptide comprises a substitution at residue DI 10. In certain embodiments, the IL-18 polypeptide comprises DI 10A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue D132. In certain embodiments, the IL- 18 polypeptide comprises DI 32A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue VI 53. In certain embodiments, the IL-18 polypeptide comprises VI 53R substitution. In certain embodiments, the IL-18 polypeptide comprises V 153E substitution. In certain embodiments, the IL-18 polypeptide comprises V153Y substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue C38. In certain embodiments, the IL-18 polypeptide comprises C38A substitution. In certain embodiments, the IL-18 polypeptide comprises C38S substitution. In certain embodiments, the IL- 18 polypeptide comprises a substitution at residue C68. In certain embodiments, the IL-18 polypeptide comprises C68A substitution. In certain embodiments, the IL-18 polypeptide comprises C68S substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue C76. In certain embodiments, the IL-18 polypeptide comprises C76A substitution. In certain embodiments, the IL-18 polypeptide comprises C76S substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue Cl 27. In certain embodiments, the IL-18 polypeptide comprises C127A substitution. In certain embodiments, the IL-18 polypeptide comprises Cl 27S substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue C38, C68, C76, and / or C127. In certain embodiments, the IL- 18 polypeptide comprises a C38A, C38S, C68A, C68S, C76A, C76S, C127A, and / or C127S substitution. In certain embodiments, the IL-18 polypeptide comprises C38A, C76A, and C127A substitutions. In certain embodiments, the IL-18 polypeptide comprises C38S, C76S and C127S substitutions.

[0240] In some embodiments, the IL-18 peptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 1, wherein the modification is E06K, VI II, K53A, S55A, or T63A. In some embodiments, the IL-18 peptide comprises at least two modifications to the amino acid sequence of SEQ ID NO: 1, wherein the modifications comprise E06K and K53A; E06K and S55A; K53 A and S55A; E06K and T63A; or K53 A and T63A. In some embodiments, the IL-18 peptide comprises at least three modifications to the amino acid sequence of SEQ ID NO: 1, wherein the modifications comprise E06K, K53A, and S55A; E06K, VI II, and K53A; E06K, C38 A, and K53 A; orE06K, K53 A, and T63 A. In some embodiments, the IL-18 peptide comprises at least four modifications to the amino acid sequence of SEQ ID NO: 1 , wherein the modifications comprise E06K, K53A, S55A, and T63A; E06K, K53A, S55A, andYOlG; E06K, K53A, S55A, and F02A; E06K, K53A, S55A, and D54A; E06K, K53A, S55A, and M51G; or C38S, C68S,WSGR Docket No. 56146-744.601C76S, and C127S. In some embodiments, the IL-18 peptide comprises at least six modifications to the amino acid sequence of SEQ ID NO: 1, wherein the modifications comprise E06K, K53A, C38S, C68S, C76S, and C127S; or K53A, T63A, C38S, C68S, C76S, and C127S. In some embodiments, the IL- 18 polypeptide comprises at least seven modifications to the sequence of SEQ ID NO: 1, wherein the seven modifications comprise E6K, VI II, C38A, K53A, T63A, C76A, Cl 27 A. In some embodiments, the IL-18 peptide comprises at least eight modifications to the amino acid sequence of SEQ ID NO: 1, wherein the modifications comprise Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A. In some embodiments, the IL-18 peptide comprises at least eight modifications to the amino acid sequence of SEQ ID NO: 1, wherein the modifications comprise Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A.

[0241] In some embodiments, IL-18 polypeptide as provided herein comprises E06Kand K53A, wherein residue position numbering of the IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL- 18 polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the IL -18 polypeptide further comprises an amino acid substitution atone or more cysteine residues. In some embodiments, the IL-18 polypeptide comprises one or more cysteines substituted with either serine or alanine. In some embodiments, the IL-18 polypeptide comprise amino acid substitutions at each cysteine residue of SEQ ID NO: 1 .

[0242] In some embodiments, the IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100 % sequence identity to SEQ ID NO: 2-72. In some embodiments, the IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100 % sequence identity to SEQ ID NO: 13 -23. In some embodiments, the IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99 % sequence identity to SEQ ID NO: 24-33 In some embodiments, the polypeptide sequence is atleast about 80% identical to SEQ ID NO: 30 or SEQ ID NO: 59. In some embodiments, the polypeptide sequence is atleast about 80% identical to SEQ ID NO: 30. In some embodiments, the polypeptidesequenceis atleast about 90% identical to SEQ ID NO: 30. In some embodiments, the polypeptidesequenceis atleast about 95% identical to SEQ ID NO: 30. In some embodiments, the polypeptidesequenceis atleast aboutWSGR Docket No. 56146-744.60198% identical to SEQ ID NO: 30 .In some embodiments, the polypeptide sequence is identical to SEQ ID NO: 30. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO: 59. In some embodiments, the polypeptide sequenceis at least about 90% identical to SEQ ID NO: 59. In some embodiments, the polypeptide sequenceis atleast about 95% identical to SEQ ID NO: 59. In some embodiments, the polypeptide sequenceis atleast about 98% identical to SEQ ID NO: 59. In some embodiments, the polypeptide sequenceis identical to SEQ ID NO: 59. In some embodiments, the IL-18 polypeptide is recombinant. In some embodiments, the IL- 18 polypeptide is one provided in Table 2. In some embodiments, the IL-18 polypeptide is one described in Table 3.Biological Activity

[0243] In some embodiments, the immunocytokine composition exhibits one or more activities associated with the antibody or antigen binding fragment and / or an IL-18 polypeptide.

[0244] In some embodiments, the immunocytokine composition exhibits an ability to bind to the IL-18 receptor. In some embodiments, the immunocytokine composition exhibits an ability to bind to the IL-18 receptor which is comparable to WT IL-18. In some embodiments, immunocytokine composition exhibits an ability to bind to the IL-18 receptor (IL-18RaP) which is reduced by at most 2-fold, at most 5-fold, at most 10-fold, at most 20-fold, at most 50-fold, at most 100-fold, at most 200-fold, at most 300-fold, at most 400-fold, or at most 1000-fold compared to WT IL-18. In some embodiments, the immunocytokine composition exhibits an enhanced ability to bind the IL-18Rap. In some embodiments, the immunocytokine composition exhibits an ability to bind to the IL-18RaP which is increased by at least 2-fold, at least 3 -fold, at least 5-fold, or at least 10-fold compared to WT IL-18.

[0245] In some embodiments, immunocytokine composition exhibits an ability to stimulate production of IFNy upon contact with a cell (e.g., an immune cell, such as an NK cell). In some embodiments, the ability of the immunocytokine composition to stimulate IFNy production is somewhat reduced compared to WT IL-18. In some embodiments, a half-maximal effective concentration (EC50) of the ability of the immunocytokine composition to stimulate production of IFNy is at most 100-fold higher than, at most 50-fold higher than, at most 20-fold higher than, at most 10-fold higher than, at most 5 -fold higher than, or at most 2-fold higher than that of a WT IL-18. In some embodiments, the ability of the immunocytokine composition to stimulate IFNy production is enhanced compared to WT IL-18. In some embodiments, a half -maximal effective concentration (EC50) of the ability of the immunocytokine composition to stimulate production of IFNy is at least 5 -fold lower than, atleast 10 -fold lower than, at least 20 -fold lower than, at leastWSGR Docket No. 56146-744.60150-fold lowerthan, atleast 75-fold lower than, or atleast 100-fold higher than that of a WT IL- 18.

[0246] In some embodiments, the immunocytokine composition exhibits an ability to stimulate production of IFNy upon contact with a cell (e.g., an immune cell, such as an NK cell) which is only somewhat reduced as compared to the IL- 18 polypeptide not comprised in the immunocytokine composition (e.g., unconjugated IL- 18 polypeptide). In some embodiments, the EC50of IFNy stimulation is at most 5-fold greaterthan, atmost 10-fold greater than, atmost 50- fold greater than, or at most 100-fold greater than that that of the IL-18 polypeptide not comprised in the immunocytokine composition. In some embodiments, the immunocytokine composition exhibits an ability to induce IFNy production in a cell as measured by half-maximal effective concentration (EC50) which is within about 100-fold of the corresponding IL-18 polypeptide not comprised in the immunocytokine composition. In some embodiments, the immunocytokine composition exhibits a lower EC50than WT IL-18. In some embodiments, the immunocytokine composition exhibits a lower EC50 than WT IL-18 by at least 2-fold, 5-fold, 10-fold, 20-fold, 30- fold, 50-fold, or 100-fold.

[0247] In some embodiments, the immunocytokine composition exhibits a reduced ability to bind IL-18 binding protein (IL-18BP). In some embodiments, the ability of immunocytokine composition to bind IL-18BP is reduced by at least 2-fold, at least 5 -fold, at least 10-fold, at least 20-fold, at least 30-fold, atleast 50-fold, or at least 100-fold compared to WT IL-18. In some embodiments, the immunocytokine composition does not display any substantial ability to bind IL- 18 BP.

[0248] In some embodiments, the immunocytokine composition exhibits a reduced ability to have its IFNy production stimulatory activity inhibited by IL-18BP. In some embodiments, the ability of the immunocytokine composition to be inhibited by IL-18BP is measured as a half maximal inhibitory concentration (IC50). In some embodiments, the immunocytokine composition exhibits an IC50 by IL-18BP that is at least 2-fold higher than, at least 5 -fold higher than, at least 10-fold higher than, at least 15 -fold higher than, at least 20-fold higher than, at least 25 -fold higher than, at least 3 O-fold higher than, at least 40-fold higher than, or atleast 50-fold higher than an IC50 of WT IL-18’s inhibition by IL-18BP. In some embodiments, the immunocytokine composition exhibits an ICso by IL-18BPthat is atleast 100-fold higher than an ICso of WT IL-18’ s inhibition by IL-18BP. In some embodiments, the immunocytokine composition exhibits an IC50 by IL- 18BPthat is at least 200-fold higher than an IC50 of WT IL-18’ s inhibition by IL-18BP. In some embodiments, the immunocytokine composition exhibits an IC50by IL-18BP thatis at least 500- fold higher than an IC50 of WT IL-18’s inhibition by IL-18BP. In some embodiments, theWSGR Docket No. 56146-744.601 immunocytokine composition exhibits an IC50 by IL-18BP that is at least 1000-fold higher than an IC50 of WT IL-18’ s inhibition by IL-18BP.

[0249] In some embodiments, the immunocytokine composition retains binding associated with the antibody or antigen binding fragment. In some embodiments, the immunocytokine composition retains binding to the antigen of the antibody or antigen binding fragment. In some embodiments, the immunocytokine composition exhibits binding affinity (KD) to the antigen of the antibody which is within 5 -fold of the binding affinity of the antibody not attached to the IL- 18 polypeptide. In some embodiments, the immunocytokine composition exhibits binding affinity (KD) to the antigen of the antibody which is within 2.5-fold of the binding affinity of the antibody not attached to the IL- 18 polypeptide. In some embodiments, the bindingis determined by ELISA. In some embodiments, the binding is determined by BLI.

[0250] In some embodiments, the immunocytokine composition retains binding to one or more Fc receptors associated with the antibody or antigen binding fragment. In some embodiments, the Fc receptor is selected from FcRn, CD64, CD32a, CD 16, and CD32b, or any combination thereof. In some embodiments, the immunocytokine composition exhibits a binding affinity (KD) to at least one Fc receptor which is within 10-fold of the binding affinity of the antibody not attached to the IL-18 polypeptide. In some embodiments, the immunocytokine composition exhibits a binding affinity (KD) to at least one Fc receptor which is less than 10 -fold higher, less than 5-fold higher, less than 4-fold higher, less than 3 -fold higher, less than 2-fold higher, or less than the binding affinity of the antibody not attached to the IL-18 polypeptide. In some embodiments, the immunocytokine composition exhibits a binding affinity (KD) to each of FcRn, CD64, CD32a, CD16, and CD32B which is less than 10-fold higher, less than 5-fold higher, less than 4-fold higher, less than 3 -fold higher, less than 2-fold higher, or less than the binding affinity of the antibody not attached to the IL-18 polypeptide. In some embodiments, the immunocytokine composition exhibits a binding affinity (KD) to each of FcRn, CD64, CD32a, CD 16, and CD32B which is within 10 -fold of the binding affinity of the antibody not attached to the IL -18 polypeptide. In some embodiments, the immunocytokine composition exhibits a binding affinity (KD) to each of FcRn, CD64, CD32a, CD16, and CD32B which is within 20-fold of the binding affinity of the antibody not attached to the IL-18 polypeptide. In some embodiments, the immunocytokine composition exhibits a binding affinity (KD) to each of FcRn, CD64, CD32a, CD16, and CD32B which is within 50-fold of the binding affinity of the antibody not attached to the IL- 18 polypeptide. In some embodiments,theimmunocytokinecompositionexhibitsabinding affinity (KD) to each of FcRn, CD64, CD32a, CD 16, and CD32B which is within 100-fold of the binding affinity of the antibody not attached to the IL- 18 polypeptide.WSGR Docket No. 56146-744.601

[0251] In some embodiments, the immunocytokine composition exhibits synergistic efficacy owing to the presence of both molecules in one molecule. In some embodiments, the immunocytokine composition exhibits enhanced activity compared to either molecule alone. In some embodiments, the immunocytokine composition exhibits enhanced anti-tumor growth inhibition compared to the antibody alone. In some embodiments, the immunocytokine composition exhibits enhanced anti -tumor growth inhibition comparedto the antibody and the IL- 18 polypeptide administered in combination. In some embodiments, the IL-18 polypeptide is administered as a half-life extended version (e.g., PEGylated, attached to anFc region (e.g., an Fc fusion), or attached to a negative control antibody). In some embodiments, the immunocytokine composition exhibits similar or enhanced antitumor activity at the same concentration as the antibody administered alone. In some embodiments, the immunocytokine composition exhibits similar or enhanced antitumor activity when administered at a dose which is less that 0.5 -fold, 0.25-fold, or 0.1 -fold the dose of the antibody alone.

[0252] In some embodiments, the immunocytokine composition (e.g., an anti-PD-1 / IL-18 immunocytokine composition) stimulates IFNy secretion by a PD-l+cell more potently thanPD- 1" cell. In some embodiments, the EC50 value is at least 5 -fold, 10-fold, 20-fold, 30 -fold, 40 -fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold lower for the PD-1+cell than the PD-1' cell. In some embodiments, the EC50 value is at least 10-fold lower for thePD-l+cell than the PD- 1- cell. In some embodiments, the EC50value is at least 50-fold lower for the PD-1+cell than the PD-1 ■ cell. In some embodiments, the EC50value is at least 100-fold lower for the PD-1+ cell than the PD-1' cell. In some embodiments, the immunocytokine composition exhibits a reduced ability to be inhibited by IL-18BP when stimulating IFNy secretion of a PD-1 + cell as compared to a PD- 1- cell. In some embodiments, the reduced ability to be inhibited by IL-18 BP is assessed as an IC50 value. In some embodiments, the IC50value is at least 10-fold, 20-fold, 50-fold, 100-fold, 150-fold, 200-fold, or 250-fold higher for the PD-1+ cell as compared to the PD- 1 ■ cell. In some embodiments, the IC50value is at least 10-fold higher for the PD-1+ cell as compared to the PD- 1" cell. In some embodiments, the IC50 value is at least 100-fold higher for the PD-1+cell as compared to the PD-L cell. In some embodiments, the IC50value is at least 200-fold higher for the PD-1+ cell as compared to the PD- 1 ' cell.

[0253] In some embodiments, the immunocytokine composition (e.g., an anti-PD-1 / IL-18 immunocytokine composition) exhibits a reduced ability to be inhibited by IL-18 BP when stimulating IFNy secretion of a PD-1+ cell compared to a corresponding IL- 18 polypeptide not comprised in an immunocytokine composition. In some embodiments, the reduced ability to be inhibited is assessed as an IC50value which is at least 2-fold, 3 -fold, 4-fold, 5-fold, 6-fold, 7-fold,WSGR Docket No. 56146-744.6018-fold, 9-fold, or 10-fold higher. In some embodiments, the reduced ability to be inhibited is assessed as an IC50 value which is at least 5 -fold higher. In some embodiments, the reduced ability to be inhibited is assessed as an IC50value which is at least 10-fold higher.

[0254] In some embodiments, an immunocytokine composition described herein (e.g., an anti- PD-1 / IL-18 immunocytokine composition) is used in a method of expanding a population of CD8+T cells in a subject in need thereof. In some embodiments, the CD8+T cells are PD-1+ / IL- 18Ra+CD8+T cells. In some embodiments, the immunocytokine composition selectively expands CD8+T cells in a tumor microenvironment relative to CD8+cells in blood of a subject. In some embodiments, the CD8+T cells are effector memory T cells. In some embodiments, the expansion of CD8+T cells is assessed as the fraction of CD8+T cells within CD45+leukocytes. In some embodiments, the fraction of CD8+T cells within CD45+leukocytes increases by at least 2 -fold, 3 -fold, or 4-fold within a tumor after administration of the immunocytokine composition. In some embodiments, the fraction of CD8+T cells within CD45+leukocytes reaches a fraction of at least 50%, 60%, 70%, 75%, or 80% within a tumor after administration of the immunocytokine composition. In some embodiments, the fraction of CD8+T cells within CD45+leukocytes increases by at most 4-fold, 3 -fold, or 2-fold in the blood of the subject after administration of the immunocytokine composition. In some embodiments, the fraction of CD8+T cells within CD45+leukocytes reaches a fraction of at most 50%, 45%, 40%, 35%, 30%, or 20% within the blood of the subject after administration of the immunocytokine composition.

[0255] In some embodiments, the immunocytokine composition (e.g., an anti-PD-1 / IL-18 immunocytokine composition) selectively suppresses macrophage cells in a tumor microenvironment relative to macrophage cells in blood of a subject. In some embodiments, suppression of macrophage cells is assessed as the fraction of macrophages within CD45+leukocytes. In some embodiments, fraction of macrophage cells within CD45 + leukocytes decreases by at least 2-fold, 3-fold, 4-fold, 5-fold, 7.5-fold, or 10-fold within a tumor after administration of the immunocytokine composition. In some embodiments, fraction of macrophage cells within CD45+ leukocytes reaches a fraction of below 40%, 30%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% within a tumor after administration of the immunocytokine composition. In some embodiments, fraction of macrophage cells within CD45+leukocytes does not have any significant effect on macrophage levels in the blood of the subject (e.g., macrophage levels remain with ~5% of the total fraction of CD45 + leukocytes in the blood).

[0256] In some embodiments, the immunocytokine composition (e.g., an anti-PD-1 / IL-18 immunocytokine composition) has minimal effect on CD4+T cells or NK cells (e.g., CD45+leukocytes) in a tumor. In some embodiments, the immunocytokine composition (e.g., an anti-WSGR Docket No. 56146-744.601PD-1 / IL- 18 immunocytokine composition) has minimal effect on the population of CD4+T cells orNK cells(e.g., CD45+leukocytes)in atumor(e.g., does not significantly expandthe population, either in absolute terms or relative to CD8+T cells). In some embodiments, the immunocytokine composition expands expression of IL- 18Ra on CD4+T cells within a tumor, optionally wherein the expression of IL-18Ra is increased by at least 2 -fold, 3-fold, or 4-fold.

[0257] In some embodiments, the immunocytokine composition (e.g., an anti-PD-1 / IL-18 immunocytokine composition) expands 1, 2, 3, or 4 cytokines selected from IFNy, GM-CSF, TNFa, and IL-6 within a tumor after administration of the immunocytokine composition. In some embodiments, the immunocytokine composition expands an amount of IFNy within a tumor after administration of the immunocytokine composition. In some embodiments, the immunocytokine composition expands an amount of GM-CSF within a tumor after administration of the immunocytokine composition. In some embodiments, the immunocytokine composition expands an amount of TNFa within a tumor after administration of the immunocytokine composition. In some embodiments, the immunocytokine composition expands an amount of IL-6 within a tumor after administration of the immunocytokine composition. In some embodiments, the immunocytokine composition does not substantially expand the cytokine in blood of a subject (e.g., each of the 1, 2, 3, or 4 cytokines). In some embodiments, the immunocytokine composition expands the cytokine (e.g., 1, 2, 3, or 4 of the cytokines) substantially more (e.g., atleast 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 1000-fold more) in the tumor of a subject than in the blood of a subject. In some embodiments, the level of the cytokine is measured at a specific amount of time after the administration (e.g., after 6h, 12h, 24h, 48h, 72h, etc.).

[0258] In some embodiments, in the immunocytokine composition preferentially expands a population of IL-18 receptor alpha / PD-1 double -positive T cells after administration in vivo (e.g, to a subject such as a human subject or a non -human primate subject). In some embodiments, the IL-18 receptor alpha / PD-1 double-positive T cells are memory T cells. In some embodiments, the IL-18 receptor alpha / PD-1 double-positive T cells are increased by a factor of at least 2 -fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more compared to those in the subject prior to the administration. In some embodiments, the IL-18 receptor alpha / PD-1 double-positive T cells reach a peak concentration after about 4 -14 days (e.g., at about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In some embodiments, the immunocytokine composition does not substantially expand non T cells which are not double positive for PD-1 and IL-18 receptor alpha, (e.g., CD3+T cells which are not double-positive for PD-1 and IL-18 receptor alpha are not preferentially expanded, only CD3+T cells which are double-positive for PD-1 and IL-18 receptor alpha are expanded).WSGR Docket No. 56146-744.601Pharmaceutical Compositions

[0259] In one aspect, provided herein is a pharmaceutical composition comprising an antibody linked to an IL-18 polypeptide described herein; and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises one or more excipients, wherein the one or more excipients include, but are not limited to, a carbohydrate, an inorganic salt, an antioxidant, a surfactant, a buffer, or any combination thereof. In some embodiments the pharmaceutical composition further comprises one, two, three, four, five, six, seven, eight, nine, ten, or more excipients, wherein the one or more excipients include, but are not limited to, a carbohydrate, an inorganic salt, an antioxidant, a surfactant, a buffer, or any combination thereof.

[0260] In some embodiments, the pharmaceutical composition further comprises a carbohydrate. In certain embodiments, the carbohydrate is selected from the group consisting of fructose, maltose, galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiose raffinose, melezitose, maltodextrins, dextrans, starches, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, cyclodextrins, and combinations thereof.

[0261] Alternately, orin addition, the pharmaceutical composition further comprises an inorganic salt. In certain embodiments, the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium sulfate, or combinations thereof.

[0262] Alternately, or in addition, the pharmaceutical composition further comprises an antioxidant. In certain embodiments, the antioxidant is selected from the group consisting of ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, potassium metabisulfite, propyl gallate, sodium metabisulfite, sodium thiosulfate, vitamin E, 3,4-dihydroxybenzoic acid, and combinations thereof.

[0263] Alternately, or in addition, the pharmaceutical composition further comprises a surfactant. In certain embodiments, the surfactant is selected from the group consisting of polysorbates, sorbitan esters, lipids, phospholipids, phosphatidylethanolamines, fatty acids, fatty acid esters, steroids, EDTA, zinc, and combinations thereof.

[0264] Alternately, or in addition, the pharmaceutical composition further comprises a buffer. In certain embodiments, the buffer is selected from the group consisting of citric acid, sodium phosphate, potassium phosphate, acetic acid, ethanolamine, histidine, amino acids, tartaric acid, succinic acid, fumaric acid, lactic acid, tris, HEPES, or combinations thereof.

[0265] In some embodiments, the pharmaceutical composition is formulated for parenteral or enteral administration. In some embodiments, the pharmaceutical composition is formulated forWSGR Docket No. 56146-744.601 intravenous (IV) or subcutaneous (SQ) administration. In some embodiments, the pharmaceutical composition is in a lyophilized form.

[0266] In one aspect, described herein is an aqueous pharmaceutical composition comprising an immunocytokine composition described herein (e.g., Composition A), and b) a buffer, a carbohydrate, and a surfactant. In some embodiments, the buffer has a pKa value of about 6. In some embodiments, the buffer comprises histidine / histidine hydrochloride. In some embodiments, the buffer is present in an amount of about 10 mM. In some embodiments, the carbohydrate comprises a sugar. In some embodiments, the sugar is sucrose. In some embodiments, the carbohydrate is present in an amount of about 280 mM. In some embodiments, the surfactant is a polysorbate. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is present in an amount of about 0.06 % (w / v). In some embodiments, the pharmaceutical composition has a pH of about 6.2. In some embodiments, the pharmaceutical composition is stored as a frozen liquid. In some embodiments, the immunocytokine composition is thawed shortly before use (e.g., less than 4 hours before, less than 2 hours before, or less than 1 hour before administration). In some embodiments, the immunocytokine composition is present in the pharmaceutical composition at a concentration of about 2 mg / mL. In some embodiments, the pharmaceutical composition is dilute in saline prior to administration.Dosage Forms

[0267] The immunocytokine compositions described herein can be in a variety of dosage forms. In some embodiments, the immunocytokine composition is dosed as a solution. In some embodiments, the immunocytokine composition is dosed as an injectable solution. In some embodiments, the immunocytokine composition is dosed as an IV solution.Methods of Treatment

[0268] In one aspect, described herein, is a method of treating cancer in a human subject in need thereof, comprising: administering to the subject an effective amount of an immunocytokine composition (z.e., an anti-PD-1 / IL-18 immunocytokine composition) or a pharmaceutical composition as described herein. In another aspect provided herein is a method of method of expanding a population of CD8+T cells in a subject in need thereof, the method comprising administering to the subject an immunocytokine composition described herein e.g., an anti-PD- 1 / IL-18 immunocytokine composition) or a pharmaceutical composition provided herein. In some embodiments, the CD8+T cells are PD-l+ / IL-18Ra+CD8+T cells. In some embodiments, the CD8+T cells are preferentially expanded in a tumor microenvironment. In some embodiments, expanding the population of CD8+T cells in the subject is useful in the treatment of cancer.WSGR Docket No. 56146-744.601

[0269] In some embodiments, the cancer is a PD-L1 positive cancer (e.g., the cancer cells express PD-L1).

[0270] In some embodiments, the cancer is a solid cancer. A cancer or tumor can be, for example, a primary cancer or tumor or a metastatic cancer or tumor. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pediatric cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cord cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureteral cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0271] In some embodiments, the cancer is resistant to treatment with at least one anti -cancer agent. In some embodiments, the cancer has been demonstrated to be resistant to at least one anticancer agent. In some embodiments, the subject as failed to respond to treatment with the at least one anti-cancer agent. In some embodiments, the subject has responded sub -optimally to treatment with the at least one anti -cancer agent (e.g., does not improve subject outcomes or extend subject life or quality of life beyond statistically significant measures, or otherwise fails to meet clinically relevant endpoints in subject). In some embodiments, the at least one anti -cancer agent is a standard of care agent for the relevant cancer type. In some embodiments, the cancer is a metastatic tumor. In some embodiments, the cancer is a refractory tumor. In some embodiments, the cancer is alocally advanced tumor. In some embodiments, the cancer is an unresectable tumor. In some embodiments, the cancer is a locally advanced unresectable tumor.

[0272] In some embodiments, the immunocytokine composition is administered after finding that the subject is non -responsive to at least one anti-cancer agent (e.g., the immunocytokine composition is used as a second-line of treatment). In some embodiments, the immunocytokine composition is administered after finding that the subject is non-responsive to at least one anticancer therapeutic regimen (e.g., a regimen comprising one or more anti -cancer agents). In some embodiments, the immunocytokine composition is administered after finding that the subject is non-responsive to a plurality of anti-cancer agents or regimens (e.g., the immunocytokine composition is administered as a third-, fourth-, or fifth-line of treatment).

[0273] In some embodiments, the cancer is resistant to treatment with an immune checkpoint inhibitor (e.g., resistant to treatment with an anti-PD-1 antibody not comprised in anWSGR Docket No. 56146-744.601 immunocytokine composition as described herein), either alone or in combination with other agents. In some embodiments, a cancer is resistant to treatment with an immune checkpoint inhibitor alone (e.g., anti-PD-1 or anti-PD-Ll antibody) when treatment with the immune checkpoint inhibitor alone is deemed clinically ineffective or sub-optimal (e.g., does not improve subject outcomes or extend subject life or quality of life beyond statistically significant measures, or otherwise fails to meet clinically relevant endpoints in subjects). In some embodiments, the cancerisresistantto treatment with an anti-PD-1 antibody alone. In some embodiments, the cancer is resistant to treatment with the checkpoint inhibitor (e.g., anti-PD-1 antibody) in combination with another active agent (e.g., an additional standard of care therapeutic). In some embodiments, treatment with the checkpoint inhibitor in combination with the other active agent offers no clinically significant benefit (i.e., administration of the checkpoint inhibitor does not improve subject outcome to a clinically significant amount as compared to the additional therapeutic alone). In some embodiments, administration of an immunocytokine composition as provided herein results in an improved subject outcome compared to a corresponding checkpoint inhibitor either alone or in combination with an additional active agent (e.g., an immunocytokine composition comprising an anti-PD-1 antibody or antigen binding fragment thereof is more efficacious than an anti-PD-1 antibody not comprised in an immunocytokine composition, either alone or in a corresponding therapy regimen with an additional active agent). In some embodiments, the cancer is resistant to treatment with an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody) after an initial response (e.g., a therapeutic response) to treatment with the immune checkpoint inhibitor (e.g., the subject initially responds well to the treatment with the immune checkpoint inhibitor, but the disease progresses at a later time period despite the initial response to the immune checkpoint inhibitor).

[0274] In some embodiments, the cancer is non-small cell lung carcinoma (NSCLC), colorectal carcinoma (CRC), or squamous cell carcinoma of the head and neck (SCCHN).

[0275] In some embodiments, the cancer is NSCLC. In some embodiments, the subject has previously receivedatleastone of a platinum-based chemotherapy, a checkpointinhibitortherapy, docetaxel, pemetrexed, or any combination thereof. In some embodiments, the subject has previously received a regiment of at least one of a platinum -based chemotherapy, a checkpoint inhibitor therapy, docetaxel, pemetrexed, or any combination thereof. In some embodiments, the subject has failed previous administration of at least one of a platinum-based chemotherapy, a checkpoint inhibitor therapy, docetaxel, pemetrexed, or any combination thereof. In some embodiments, the cancer is resistant or refractory to the prior treatment with the platinum -based chemotherapy, checkpoint inhibitor therapy, docetaxel, pemetrexed, or any combination thereof.WSGR Docket No. 56146-744.601In some embodiments, the prior therapy was the checkpoint inhibitor therapy. In some embodiments, the checkpoint inhibitor therapy was an anti-PD-1 or anti-PD-Ll antibody therapy. In some embodiments, the NSCLC lacks a sensitizing mutation (i.e., a mutation which renders the NSCLC sensitive to another therapeutic intervention). In some embodiments, the NSCLC lacks a sensitizing mutation in EGFR, ALK, ROS1, BRAF, MET (e.g., METexl4 skipping), RET, ERBB2, NTRK, or any combination thereof. In some embodiments, the NSCLC lacks a sensitizing mutation in any one of EGFR, ALK, ROS1, BRAF, MET (e.g., METexl4 skipping), RET, ERBB2 and NTRK.

[0276] In some embodiments, the cancer is CRC. In some embodiments, the CRC is micro satellite stable and / or proficient mismatch repair. In some embodiments, the CRC is recurrent or intolerant after one or more prior regimen of chemotherapy. In some embodiments, the prior regimen of chemotherapy comprises one or more of a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), oxaliplatin, irinotecan, an EGFR inhibitor (e.g., cetuximab, panitumumab, etc.), a VEGF / VEGFR2 pathway inhibitor (e.g., bevacizumab, ziv-aflibercept, ramucirumab, etc.), a thymidine phosphorylase inhibitor / fluoropyrimidine combination therapy (e.g., tipiricil / trifluridine), an anti-HER2 agent, a multikinase inhibitor (e.g., regorafenib, fruquintinib, etc.), a BRAF inhibitor (e.g., encorafinib), an immune checkpoint inhibitor, or any combination thereof. In some embodiments, the prior regimen of chemotherapy comprises a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), oxaliplatin and irinotecan. In some embodiments, the prior regimen of chemotherapy comprises a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), oxaliplatin, irinotecan and a VEGF / VEGFR2 pathway inhibitor (e.g., bevacizumab, ziv- aflibercept, ramucirumab, etc.). In some embodiments, the prior regimen of chemotherapy comprises a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), oxaliplatin, irinotecan and bevacizumab. In some embodiments, the priorregimen of chemotherapy comprises an anti-EGFR agent (e.g., for CRC having wild type KRAS / NRAS / BRAF). In some embodiments, the prior regimen of chemotherapy comprisesan anti-HER2 antibody or antigen binding fragment thereof (e.g., for HER-2 positive CRC). In some embodiments, the prior regimen of chemotherapy comprises a BRAF inhibitor (e.g., encorafenib) (e.g., for CRC having a BRAF V600E mutation). In some embodiments, the prior chemotherapy regimen comprises regorafenib, trifluridine / tripiracil, or fruquintinib. In some embodiments, the CRC is KRAS, NRAS, and / or BRAF wild type CRC. In some embodiments, the CRC is KRAS, NRAS, and BRAF wild type CRC.

[0277] In some embodiments, the cancer is SCCHN. In some embodiments, the subject has not received an immune checkpoint inhibitor therapy (e.g., an anti-PDl, anti-PD-Ll, etc.). In someWSGR Docket No. 56146-744.601 embodiments, the subject is refractory or otherwise unresponsive to treatment with a previous therapeutic intervention. In some embodiments the subject has previously received a platinumbased chemotherapy (e.g., cisplatin, carboplatin, etc.), a checkpoint inhibitor therapy, a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), or any combination thereof. In some embodiments, the subject has previously received radiation therapy. In some embodiments, the subject has previously received an anti-EGFR antibody (e.g., Cetuximab), optionally in combination with radiotherapy. In some embodiments, the subject has previously received cisplatin or carboplatin, a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), and an anti- PD-1 antibody (e.g., Pembrolizumab) in combination. In some embodiments, the subject has previously received cisplatin and an anti-PD-1 antibody (e.g., Pembrolizumab) in combination. In some embodiments, the subject has previously received carboplatin and an anti-PD-1 antibody (e.g., Pembrolizumab) in combination. In some embodiments, the subject has previously received cisplatin, a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), and an anti-PD-1 antibody (e.g., Pembrolizumab) in combination. In some embodiments, the subject has previously received carboplatin, a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), and an anti-PD-1 antibody (e.g., Pembrolizumab) in combination. In some embodiments, the subject has previously received a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.) and an anti-PD-1 antibody (e.g., Pembrolizumab) in combination.

[0278] In some embodiments, the immunocytokine composition is administered in a dose of from about from about 1 microgram / kg to about 1000 microgram / kg. In some embodiments, the immunocytokine composition is administered in a dose of from about from about 1 microgram / kg to about 1000 microgram / kg per administration. In some embodiments, the immunocytokine composition is administered at a dose of about 3 microgram / kg, about 10 microgram / kg, about 30 microgram / kg, about 100 microgram / kg, about 300 microgram / kg, about450 microgram / kg, or about 600 microgram / kg. In some embodiments, the immunocytokine composition is administered at a dose of about 3 microgram / kg, about 10 microgram / kg, about 30 microgram / kg about 100 microgram / kg, about 300 microgram / kg, about 450 microgram / kg, or about 600 microgram / kg per administration. In some embodiments, the immunocytokine composition is administered at a dose of about 100 microgram / kg. In some embodiments, the immunocytokine composition is administered at a dose of about 100 microgram / kg per administration. In some embodiments, the immunocytokine composition is administered at a dose of about 200 microgram / kg. In some embodiments, the immunocytokine composition is administered at a dose of about 200 microgram / kg per administration. In some embodiments, the immunocytokine composition is administered at a dose of about 300 microgram / kg. In some embodiments, theWSGR Docket No. 56146-744.601 immunocytokine composition is administered at a dose of about 300 microgram / kg per administration. In some embodiments, the dose of immunocytokine composition administered is less than that required for a corresponding antibody (e.g., a PD-1 antibody).

[0279] In some embodiments, the dose (e.g., one of the doses described herein) produces a Cmax in the subject of at least about 1000 ng / mL, 2000 ng / mL, 3000 ng / mL, 4000 ng / mL, or 5000 ng / mL. In some embodiments, the dose produces a Cmax in the subject of at most about 100,000 ng / mL, 90,000 ng / mL, 80,000 ng / mL, 70,000 ng / mL, 60,000 ng / mL, 50,000 ng / mL, 40,000 ng / mL, 30,000 ng / mL, 25,000 ng / mL, 20,000 ng / mL, 15,000 ng / mL, or 10,000 ng / mL. In some embodiments, the dose produces an AUC(O-inf) in the subject of atleast about 10,000 h*ng / mL, 20,000 h*ng / mL, 50,000h*ng / mL, 75,000h*ng / mL, or 100,000 h*ng / mL. In some embodiments, the dose produces an AUC(O-inf) in the subject of at most about 1,000,000 h* ng / mL, 750,000 h*ng / mL, 500,000 h*ng / mL, 450,000 h*ng / mL, 400,000 h*ng / mL, 350,000 h*ng / mL, 300,000 h*ng / mL, 275,000 h*ng / mL, 250,000 h*ng / mL, 225,000 h*ng / mL, 200,000 h*ng / mL, 175,000 h*ng / mL, or 150,000 h*ng / mL.

[0280] In some embodiments, the method of treating cancer comprises administering multiple doses of the immunocytokine composition to the subject. In some embodiments, the multiple doses comprise at least 2, 3, 4, 5, or 6 doses. In some embodiments, doses are administered about every 2 weeks. In some embodiments, doses are administered about every 2 weeks for a period of at least 2 weeks, at least one month, at least 2 months, at least 4 months, at least 6 months, or at least 1 year.

[0281] Combination therapies with one or more additional active agents are contemplated herein.

[0282] An effective response is achieved when the subject experiences partial or total alleviation or reduction of signs or symptoms of illness, and specifically includes, without limitation, prolongation of survival. The expected progress! on -free survival times may be measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors. Prolonging survival includes without limitation times of atleast 1 month (mo), about at least 2 mos., about at least 3 mos., about at least 4 mos., about at least 6 mos., about at least 1 year, about at least 2 years, about at least 3 years, about at least 4 years, about at least 5 years, etc. Overall or progression-free survival can be also measured in months to years. Alternatively, an effective response may be that a subject’s symptoms or cancer burden remain static and do not worsen. Further treatment of indications are described in more detail elsewhere herein. In some instances, a cancer or tumor is reduced by at least 2%, 5%, 10%, 15%,WSGR Docket No. 56146-744.60120%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0283] In some embodiments, the immunocytokine composition is administered in a single dose of the effective amount of immunocytokine composition, including further embodiments in which (i) the immunocytokine composition is administered once a day. In some embodiments, the immunocytokine composition is administered periodically (e.g., multiple doses are administered at different time points). In some embodiments, the immunocytokine composition is administered about every 2 weeks. Administration includes, but is not limited to, injection by any suitable route (e.g., parenteral, enteral, intravenous, subcutaneous, etc.). In some embodiments herein, the administration is performed intravenously.Methods of Manufacturing

[0284] In one aspect, described herein, is a method of making an immunocytokine composition, comprisingproviding an antibody or antigen bindingfragmentthereof e.g., an antibody or antigen binding fragment provided herein), wherein the antibody comprises a reactive group (e.g., a conjugation handle), contacting the reactive group with a complementary reactive group attached to an IL-18 polypeptide, and forming the immunocytokine composition. The resulting composition is any of the compositions provided herein.

[0285] In some embodiments, providing the antibody comprising the reactive group comprises attaching the reactive group to the antibody. In some embodiments, the reactive group is added site-specifically. In some embodiments, attaching the reactive group to the antibody comprises contacting the antibody with an affinity group comprising a reactive functionality which forms a bond with a specific residue of the antibody. In some embodiments, attaching the reactive group to the antibody comprises contacting the antibody with an enzyme. In some embodiments, the enzyme is configured to site-specifically attach the reactive group to a specific residue of the antibody. In some embodiments, the enzyme is glycosylation enzyme or a transglutaminase enzyme.

[0286] In some embodiments, the method further comprises attachingthe complementary reactive group to the cytokine.Definitions

[0287] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.WSGR Docket No. 56146-744.601

[0288] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0289] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0290] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0291] The term “about” or “approximately” can mean within an acceptable error range for the particularvalue as determined by one of ordinary skill in the art, which will depend in parton how the value is measured or determined, i.e. , the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5 -fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0292] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusiveor open- ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.WSGR Docket No. 56146-744.601

[0293] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” meansthat a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0294] Referred to herein are groups which are “attached” or “covalently attached” to residues of IL-18 polypeptides or other polypeptides. As used herein, “attached” or “covalently attached” means that the group is tethered to the indicated reside, and such tethering can include a linking group (i.e., a linker). Thus, for a group “attached” or “covalently attached” to a residue, it is expressly contemplated that such linking groups are also encompassed.

[0295] Binding affinity refers to the strength of a binding interaction between a single molecule and its ligand / binding partner. A higher binding affinity refers to a higher strength bond than a lower binding affinity . In some instances, binding affinity is measuredby the dissociation constant (KD) between the two relevant molecules. When comparing KDvalues, a binding interaction with a lower value will have a higher binding affinity than a binding interaction with a higher value. For a protein-ligand interaction, KDis calculated according to the following formula:where [L] is the concentration of the ligand, [P] is the concentration of the protein, and [LP] is the concentration of the ligand / protein complex.

[0296] Referred to herein are certain amino acid sequences (e.g., polypeptide sequences) which have a certain percent sequence identity to a reference sequence or refer to a residue at a position corresponding to a position of a reference sequence. Sequence identity is measured by protein - protein BLAST algorithm using parameters of Matrix BLOSUM62, Gap Costs Existence! 1, Extension!, and Compositional Adjustments Conditional Compositional Score Matrix Adjustment. This alignment algorithm is also used to assess if a residue is at a “corresponding” position through an analysis of the alignment of the two sequences being compared.

[0297] The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia (U.S.P.) or other generally recognized pharmacopeia for use in animals, including humans.

[0298] A “pharmaceutically acceptable excipient, carrier, or diluent” refers to an excipient, carrier, or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.WSGR Docket No. 56146-744.601

[0299] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub -range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 .8, and 1 .9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0300] Throughout the instant description, certain numerical or other similar values may be described as, for example, “at least” or “at most” a set of values indicated in a list form (e.g., “at least 2, 3, 4, 5, or 6”). In such cases, unless context clearly indicates otherwise, it is intended that the phrase “at least,” “at most,” or other similar term is applied individually to each value in the list. For example, the phrase “atleast2, 3, 4, 5, or 6” is to be interpreted as “at least2, at least 3, at least 4, at least 5, or at least 6.”

[0301] Certain formulas and other illustrations provided herein depict triazole reaction products resulting from azide-alkyne cycloaddition reactions. While such formulas generally depict only a single regioisomer of the resulting triazole formed in the reaction, it is intended that the formulas encompass both resulting regioisomers. Thus, while the formulas depict only a single regioisomer(e.g.), t s nten e t at t e ot er reg o somer (e.g. is also encompassed.

[0302] The term “subject” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non -human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.

[0303] The term “optional” or “optionally” denotes that a subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.

[0304] The term “moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.WSGR Docket No. 56146-744.601

[0305] As used herein, “conjugation handle” refers to a reactive group capable of forming a bond upon contacting a complementary reactive group. In some instances, a conjugation handle preferably does not have a substantial reactivity with other molecules which d o not comprise the intended complementary reactive group. Non-limiting examples of conjugation handles, their respective complementary conjugation handles, andcorrespondingreactionproducts can be found in the table below. While table headings place certain reactive groups under the title “conjugation handle” or “complementary conjugation handle,” it is intended that any reference to a conjugation handle can instead encompass the complementary conjugation handles listed in the table (e.g., a trans-cyclooctene can be a conjugation handle, in which case tetrazine would be the complementary conjugation handle). In some instances, amine conjugation handles and conjugation handles complementary to amines are less preferable for use in biological systems owing to the ubiquitous presence of amines in biological systemsand the increased likelihood for off-target conjugation.Table of Conjugation Handles

[0306] Through outthe instant application, prefixes are used before the term “conjugationhandle” to denote the functionality to which the conjugation handle is linked. For example, a “protein conjugation handle” is a conjugation handle attached to a protein (either directly or through a linker), an “antibody conjugation handle” is a conjugation handle attached to an antibody (either directly or through a linker), and a “linker conjugation handle” is a conjugation handle attached to a linker group (e.g., a bifunctional linker used to link a synthetic protein and an antibody).WSGR Docket No. 56146-744.601

[0307] The term “alkyl” refers to a straight or branched hydrocarbon chain radical, having from one to twenty carbon atoms, and which is attached to the rest of the molecule by a single bond. An alkyl comprising up to 10 carbon atoms is referred to as a C1-C10 alkyl, likewise, for example, an alkyl comprising up to 6 carbon atoms is a Ci-C6alkyl. Alkyls (and other moieties defined herein) comprising other numbers of carbon atoms are represented similarly. Alkyl groups include, but are not limited to, Ci-Cio alkyl, C1-C9 alkyl, Ci-C8alkyl, C1-C7 alkyl, Ci-C6alkyl, Ci- C5alkyl, C C4alkyl, - alkyl, C C2alkyl, C2-C8alkyl, C3-C8alkyl and C4-C8alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, zz-propyl, 1 -methyl ethyl (z-propyl), / / -butyl, z-butyl, 5 -butyl, zz-pentyl, 1,1 -dimethyl ethyl (z-butyl), 3 -methylhexyl, 2- methylhexyl, 1 -ethyl-propyl, and the like. In some embodiments, the alkyl is methyl or ethyl. In some embodiments, the alkyl is -CH(CH3)2or -C(CH3)3. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted. “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group. In some embodiments, the alkyleneis -CFF-, -CH2CH2-, or-CH2CH2CH2-. In some embodiments, the alkylene is -CH2-. In some embodiments, the alkylene is -CH2CH2-. In some embodiments, the alkylene is -CH2CH2CH2-. Unless stated otherwise specifically in the specification, an alkylene group may be optionally substituted.

[0308] The term “alkenylene” or “alkenylene chain” refers to a straight or branched divalent hydrocarbon chain in which at least one carbon-carbon double bond is present linking the rest of the molecule to a radical group. In some embodiments, the alkenylene is -CH=CH-, -CH2CH=CH- , or -CH=CHCH2-. In some embodiments, the alkenylene is -CH=CH-. In some embodiments, the alkenylene is -CH2CH=CH-. In some embodiments, the alkenylene is -CH=CHCH2-.

[0309] The term “alkynyl” refers to a type of alkyl group in which at least one carbon -carbon triple bond is present. In one embodiment, an alkenyl group has the formula -C=C-RX, wherein Rxrefers to the remaining portions of the alkynyl group. In some embodiments, Rxis H or an alkyl. In some embodiments, an alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Non -limiting examples of an alkynyl group include -C=CH, -C=CCH3, - C=CCH2CH , and -CH2C=CH.

[0310] The term “aryl” refers to a radical comprising at least one aromatic ring wherein each of the atoms forming the ring is a carbon atom. Aryl groups can be optionally substituted. Examples of aryl groups include, but are not limited to phenyl, and naphthyl. In some embodiments, the aryl is phenyl. Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylene group). Unless stated otherwise specifically in the specification, the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meantto include aryl radicals that are optionally substituted.WSGR Docket No. 56146-744.601In some embodiments, an aryl group comprises a partially reduced cycloalkyl group defined herein (e.g., 1,2-dihydronaphthalene). In some embodiments, an aryl group comprises a fully reduced cycloalkyl group defined herein (e.g., 1,2, 3, 4 -tetrahydronaphthalene). When aryl comprises a cycloalkyl group, the aryl is bonded to the rest of the molecule through an aromatic ring carbon atom. An aryl radical can be a monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) ring system, which may include fused, spiro or bridged ring systems.

[0311] The term “cycloalkyl” refers to a monocyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In some embodiments, cycloalkyls are saturated or partially unsaturated. In some embodiments, cycloalkyls are spirocyclic or bridged compounds. In some embodiments, cycloalkyls are fused with an aromatic ring (in which case the cycloalkyl is bonded through a non -aromatic ring carb on atom). Cycloalkyl groups include groups having from 3 to 10 ring atoms. Representative cycloalkyls include, but are notlimitedto, cycloalkyls havingfromthreeto ten carbon atoms, from three to eight carbon atoms, from three to six carbon atoms, or from three to five carbon atoms. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl is cyclopentyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl. Polycyclic radicals include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetrainyl, decalinyl, 3,4- dihydronaphthalenyl-l(2H)-one, spiro[2.2]pentyl, norbomyl and bicyclefl. l.l]pentyl. Unless otherwise stated specifically in the specification, a cycloalkyl group maybe optionally substituted.

[0312] The term “heteroalkylene” or “heteroalkylene chain” refers to a straight or branched divalent heteroalkyl chain linking the rest of the molecule to a radical group. Unless stated otherwise specifically in the specification, the heteroalkyl or heteroalkylene group may be optionally substituted as described below. Representative heteroalkylene groups include, but are not limited to -CH2-O-CH2-, -CH2-N(alkyl)-CH2-, -CH2-N(aryl)-CH2-, -OCH2CH2O-, - OCH2CH20CH2CH2O-, or - O CH2CH2O CH2CH2O CH2CH2O -.

[0313] The term “heteocycloalkyl” refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless stated otherwise specifically in the specification, the heterocycloalkyl radical may be a monocyclic, or bicyclic ring system, which may include fused (when fused with an aryl or a heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized. The nitrogen atom may be optionally quaternized. The heterocycloalkyl radical is partially or fully saturated. Examples ofWSGR Docket No. 56146-744.601 heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1 -oxo- thiomorpholinyl, 1, 1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. Unless otherwise noted, heterocycloalkyls have from 2 to 12 carbons in the ring In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 1 or 2N atoms. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 3 or 4 N atoms. In some embodiments, heterocycloalkyls have from 2 to 12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring. In some embodiments, heterocycloalkyls have from 2 to 12 carbons, 1 -3 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. It is understoodthat when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring). Unless stated otherwise specifically in the specification, a heterocycloalkyl group may be optionally substituted.

[0314] The term “heteroaryl” refers to an aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, heteroaryl is monocyclic or bicyclic. Illustrative examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8 -naphthyridine, and pteridine. Illustrative examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Illustrative examples of bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl or furyl. In some embodiments, a heteroaryl contains 0-6 N atoms in the ring. In some embodiments, a heteroarylWSGR Docket No. 56146-744.601 contains 1-4 N atoms in the ring. In some embodiments, a heteroaryl contains 4 -6 N atoms in the ring. In some embodiments, a heteroaryl contains 0-4 N atoms, 0-1 0 atoms, 0-1 P atoms, and 0- 1 S atoms in the ring. In some embodiments, a heteroaryl contains 1-4 N atoms, 0-1 0 atoms, and 0-1 S atoms in the ring. In some embodiments, heteroaryl is a C1-C9 heteroaryl. In some embodiments, monocyclic heteroaryl is a C1-C5 heteroaryl. In some embodiments, monocyclic heteroaryl is a 5 -membered or 6-membered heteroaryl. In some embodiments, a bicyclic heteroaryl is a C6-C9 heteroaryl. In some embodiments, a heteroaryl group comprises a partially reduced cycloalkyl or heterocycloalkyl group defined herein (e.g., 7,8 -dihydroquinoline). In some embodiments, a heteroaryl group comprises a fully reduced cycloalkyl or heterocycloalkyl group defined herein (e.g., 5,6,7, 8 -tetrahydroquinoline). When heteroaryl comprises a cycloalkyl or heterocycloalkyl group, the heteroaryl is bonded to the rest of the molecule through a heteroaromatic ring carbon or hetero atom. A heteroaryl radical can be a monocyclic or polycyclic (e.g., bicyclic, tricyclic, ortetracyclic) ring system, which may include fused, spiro or bridged ring systems.

[0315] The term “optionally substituted” or “substituted” means that the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from D, halogen, -CN, -NH2, -NH(alkyl), -N(alkyl)2, -OH, -CO2H, -CO2alkyl, - C(=0)NH2, -C(=0)NH(alkyl), -C(=0)N(alkyl)2, -S(=0)2NH2, -S(=0)2NH(alkyl), -S(=0)2N(alkyl)2, alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone. In some other embodiments, optional substituents are independently selected fromD, halogen, -CN, -NH2, -NH(CH3), -N(CH3)2, -OH, -C02H, -C02(Ci-C4alkyl), - C(=0)NH2, - C(=0)NH(C 1 -C4alkyl), -C(=0)N(Ci-C4alkyl)2, -S(=0)2NH2, -S(=0)2NH(Ci- C4alkyl), - S(=0)2N(Ci-C4alkyl)2, Ci-C4alkyl, C3-Cecycloalkyl, Ci-C4fluoroalkyl, Ci-C4heteroalkyl, C4- C4alkoxy, Ci-C4fluoroalkoxy, -SCi-C4alkyl, -S(=0)Ci-C4alkyl, and -S(=0)2Ci- C4alkyl. In some embodiments, optional substituents are independently selected from D, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -NH(cyclopropyl), -CH3, -CH2CH3, -CF3, -OCH3, and - OCF3. In some embodiments, substituted groups are substituted with one or two of the preceding groups. In some embodiments, an optional substituent on an aliphatic carbon atom (acyclic or cyclic) includes oxo (=0).

[0316] As used herein, “AJICAP™ technology,” “AJICAP™ methods,” and similar terms refer to systems and methods (currently produced by Ajinomoto Bio-Pharma Services (“Ajinomoto”)) for the site specific functionalization of antibodies and related molecules using affinity peptides to deliver the desired functionalization to the desired site. General protocols for the AJICAP™WSGR Docket No. 56146-744.601 methodology are found at least in PCT Publication No. WO2018199337A1, PCT Publication No. WO20 19240288 Al, PCT Publication No. WO2019240287A1, PCT Publication No. W02020090979A1, Matsuda et al., Mol. Pharmaceutics 2021, 18, 4058-4066, and Yamada et al., AJICAP: Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies. Ari ew. Chem., Int. Ed. 2019, 58, 5592-5597, and in particular Examples 2-4 of US Patent Publication No. US20200190165A1 . In some embodiments, such methodologies site specifically incorporate the desired functionalization at lysine residues at a position selected from position 246, position 248, position 288, position 290, and position 317 of an antibody Fc region (e.g., an IgGl Fc region) (EU numbering). In some embodiments, the desired functionalization is incorporated at residue position 248 of an antibody Fc region (EU numbering). In some embodiments, position 248 corresponds to the 18thresidue in a human IgG CH2 region (EU numbering).

[0317] As used herein, “Composition A” refers to an immunocytokine comprised of anti-PD-1 antibody LZM-009 conjugated to a single IL-18 polypeptide of SEQ ID NO: 30 using AJICAP™ technology. The IL-18 polypeptide is attached to LZM-009 atthe K248 residue of the Fc domain via a linker of the structurewhich is also attached to residue C68 of the IL-18 polypeptide (i.e., the left side of the linker above is attached to the Fc domain and the right side of the linker above is attached to C68) . As is apparent to one of ordinary skill in the art, the triazole portion of the linker can also be the regioisomer of the azide-alkyne reaction, and the actual product Composition A will exist as a mixture of these regioisomers.Sequences (SEQ ID NOS) of IL-18 PolypeptidesTABLE 2WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601WSGR Docket No. 56146-744.601ADDITIONAL EXEMPLARY IL-18 CONSTRUCTS

[0318] Also provided herein are IL-18 polypeptides which comprise the modifications to SEQ ID NO: 1 listed in the table below, each of which is assigned a Composition ID, which can be incorporated into an immunocytokine composition as provided herein. In some emb odiments, the IL- 18 polypeptide of an immunocytokine composition comprises the set of amino acid substitutions shown for any oneof the constructs depictedbelow. In the constructs depicted below, each of the substitutions is listed using SEQ ID NO: 1 as a reference sequence. In some embodiments, the IL-18 polypeptide an immunocytokine composition comprises only the substitutions shown for a construct below relative to SEQ ID NO: 1 (i.e., the IL- 18 polypeptideWSGR Docket No. 56146-744.601 has only the indicated set of substitutions and the remaining residues are those set forth in SEQID NO: 1).Table 3 - Additional IL-18 PolypeptideWSGR Docket No. 56146-744.601

[0319] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosure as defined in the appended claims.

[0320] The present disclosure is further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the disclosure in any way.EXAMPLESExample 1A - Recombinant IL18 Expression and Purification

[0321] RecombinantIL-18 variants suitable for linking to an antibody or antigen bindingfragment as provided herein can be prepared according to the protocols provided below. In some instances, the recombinant IL- 18 will contain a cysteine residue at the desired point of attachment of the linker, or may include an unnatural amino acid (e.g., azidolysine) suitable for attachment of the linker at the desired point of attachment.Soluble His-SUMO-IL18 variants

[0322] E. colt BL21 (DE3) harboring a plasmid encoding a N-His-SUMO tagged IL-18 variant fusion is inoculated into 3 L LB culture medium and induced with 0.4 mM IPTG at 30 °C for 6h. Cells are pelleted and cell lysis is done by sonication in lysis buffer: PBS, pH 7.4. Soluble protein is purified viaNi-NTAbeads6FF (wash 1 with: PBS, 20 mM imidazole, pH7.4; wash 2 with PBS, 50 mM Imidazole, pH7.4; elution with PBS, 500 mM imidazole, pH7.4).

[0323] Fractions containing the protein are pooled, dialyzed into PBS pH 7.4 and followed by SUMO digestion. Then the protein is two-step purified with Ni-NTA beads (continue with flow through sample) and gel filtration. Fractions containing the protein are pooled and QC is performed using analytical techniques, such as SDS-PAGE and analytical SEC.Insoluble His-SUMO-IL18 variants

[0324] E. co / z' BL21 (DE3) harboring a plasmid encoding a N-His-SUMO tagged IL-18 variant fusion are inoculated into 10 L LB culture medium and induced with 0.4 mMIPTG at 30 °C forWSGR Docket No. 56146-744.6016h. Cells are pelleted and cell lysis is done by sonication in lysis buffer: PBS, 8 M urea, pH 7.4. Protein is purified viaNi-NTA beads 6FF (wash 1 with: PBS, 8 M urea, 20 mM imidazole, pH7.4; wash 2 with PBS, 8 M urea, 50 mM Imidazole, pH7.4; elution with PBS, 8 M urea, 500 mM imidazole, pH7.4).

[0325] Fractions containing the protein are pooled, dialyzed into PBS pH 7.4 and followed by SUMO digestion. Then the protein is purified with Ni-NTA beads (equilibrate column with PBS, 8 M urea, pH 7.4, wash with PBS, 8 M urea, pH 7.4, elution with PBS, 8 M urea, pH 7.4). Fractions containing the protein are pooled, dialyzed into PBS pH 7.4 and QC is performed using analytical techniques, such as SDS-PAGE and analytical SEC.Insoluble tagless IL18 variants

[0326] E. coliB JA (DE3) harboring a plasmid encoding mlL- 18 is inoculated into 2 L LB culture medium and induced with 0.4 mM IPTG at 30 °C for 6h. Cells are pelleted and cell lysis was done by sonication in lysis buffer: 1 lO mM Tris, 1.1 M guanidine HC1, 5 mMDTT, pH 8.9. Protein as purified via Q Sepharose FF (balance buffer 20 mM MES, pH 7.0, elution with an increasing gradient from 0 to 1 M NaCl).Example IB -Additional Methods for Recombinant IL18 Expression and Purification

[0327] The following protocols were also used to prepare certain IL-18 polypeptides provided herein which were subsequently used either in assays for conversion into immunocytokine compositions as provided herein.

[0328] Expression of IL-18 Polypeptides

[0329] IL- 18 polypeptide were produced as an N -terminal fusion to N -His-SUMO-IL 18. The gene was synthesized and cloned by a commercial vendor. Plasmids were transformed into E. coliBLZ (DE3). Expression was performed in shake flasks with TB medium. The cells were grown at 37 °C until an OD600 of approximately 1.2 was reached, after which they were inducedby 0.1 mM IPTG and cultured for another 20 hours at 18 °C. Cells were harvested by centrifugation.

[0330] Purification of IL18 Polypeptides

[0331] Cell lysis - Cells were resuspended in lysis buffer (20 mM Tris / HCl, pH 8.0, 0.15 MNaCl, 10 mM Imidazole, 1 tablet of EDTA-free complete protease inhibitor (Roche, COEDTAF-RO) per liter production) at 100 mL buffer / L culture and disrupted twice with a homogenizer at 1000 bar. The lysate was cleared of debris by centrifugation at 40’000 g for 2x 45 minutes, changing flask in between, and subsequent filtration through a 0.22 pm filter.

[0332] Affinity Purification and Endotoxin Removal - The lysate was loaded on Ni NTA resin (Cytiva, 17524802) pre-equilibrated with 20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM Imidazole, at 5 mL / min and washed with the same buffer for 5 CV. To remove endotoxins, theWSGR Docket No. 56146-744.601 column was washed with 20 mM Tris / HCl, pH 8.0, 0. 15 MNaCl, 10 mM Imidazole, 0.1% Tryton X-l 14 at 10 mL / min for 30 CV. The column was washed with 20 mM Tris / HCl, pH 8.0, 0. 15 M NaCl, 10 mM Imidazole, for 5 CV at 5 mL / min and the protein of interest eluted by linear increase of imidazole concentration. The column was then regenerated by 0.5M NaOH.

[0333] SUMO digestion and dialysis - To cleave the SUMO tag, SUMO protease was added to the elution pool at a w / w ratio of 1 :250 (protein:SUMO enzyme) and incubated for 18 hours at 4°C. At the same time, the protein was dialysed (20 mM Tris, pH 8.0, 150mMNaCl), to reduce the imidazole concentration.

[0334] Purification by reverse IMAC -In orderto remove the cleaved tag and the SUMO protease, the digested protein was flown through a Ni NTA resin column pre -equilibrated with 20 mM Tris / HCl, pH 8.0, 0.15 MNaCl, 10 mM Imidazole, at 5 mL / min. The flow-through was collected.

[0335] Buffer Exchange - The flow-through was concentratedto 2.6 mg / mL and buffer exchanged into either 20mM HEPES, 150mM NaCl, 0.5mM TCEP, 10% glycerol, pH7.5 or PBS, 10% glycerol, pH7.4. Proteins were stored at -70°C until further quality controls.Example 2 - Conjugation of IL-18 Polypeptide With Bifunctional Linking Group

[0336] An IL-18 polypeptide as provided herein can be conjugated to a bifunctional linkinggroup prior to forming the full linker of the immunocytokine composition. In some cases, the bifunctional linkinggroup first attaches to a desired residue of the IL-18 polypeptide at the point of attachment of the linker. Once attached to the IL-18 polypeptide, the second functionality of the bifunctional linking group is used to attach to a second portion. An exemplary schematic of such a process is shown in FIGURE 6. The process shown in FIGURE 6 is not needed for all IL- 18 polypeptides, as some IL-18 polypeptides provided herein (e.g., an IL-18 with the desired conjugation handle for final linkage with the antibody already attached). An exemplary protocol on an IL-18 polypeptide with a cysteine residue point of attachment provided herein is described below.

[0337] Conjugation - The IL-18 polypeptide is stored at a concentration of 2.4 mg / mL at -80 °C in potassium phosphate buffer (pH 7.0) containing 50 mM KC1 and 1 mM DTT. The sample is thawed on ice yielding a clear solution. The protein solution is diluted in PBS, pH 7.4. A clear solution is obtained at a concentration of ~ 0.4 mg / mL.

[0338] The protein solution is dialyzed against PBS, pH 7.4 (twice against 600 mL for 2 h and once against 800 mL for 18 h). After dialysis, a clear solution is obtained with no sign of precipitation. Protein concentration is obtained using UV absorbance at 280 nm and by BCA protein assay.WSGR Docket No. 56146-744.601

[0339] A stock solution of bi-functional linking group (e.g., bromoacetamido-PEG5-azide, CAS: 1415800-37-1) in water is prepared at a concentration of 20 mM. 500 pL of the protein solution are mixed with 25 pL of linking group solution. pH was adjusted to 7.5 and it was let to react for 3 h at 20 °C.

[0340] The progress of the synthesis is monitored by reverse-phase HPLC using a gradient of 5 to 30% (2.5 min) and 30 to 75% (7.5 min) CH3CN with 0.1% TFA (v / v) on a Aeris WIDEPORE Cl 8 200 A column (3.6 pm, 150 x 4.6 mm) at a flow rate of 1 mL / min at 40 °C and by MALDI- TOF MS.

[0341] Purification - In some cases, ion-exchange chromatography is used to purify the conjugated protein. To remove the excess of probe, the reaction mixture (volume is around 500 pL) is flowed through aHi-Trap-G-FF-1 mL column using25 mM Tris (pH 7.4) as the buffer. The column is eluted with a linear gradient of 0-0.35 M NaCl in the same buffer. The fractions containing the target protein are gathered, buffer exchanged (25 mM Tris, pH 7.4, 75 mM NaCl, 5% glycerol) and concentrated at 0.4 mg / mL. The concentration of purified protein is determined by UV absorbance at 280 nm and by BCA protein assay. The protein solution is kept at -80 °C

[0342] Characterization - The purity and identity of the recombinant protein from commercial source and the conjugated protein is confirmed by aSEC, HPLC and MALDI-TOF MS.Example 3 - Conjugation of IL-18 Polypeptide to Antibody

[0343] Preparation of a conjugatable antibody

[0344] A modified antibody (e.g., an anti-PD-1 antibody such as nivolumab or LZM-009) comprising a DBCO conjugation handle is prepared using a protocol modified from Examples 2- 4 of US Patent Publication No. US2020019165A1. An exemplary illustration of this process resulting in the attachment of one DBCO conjugation handle is shown in FIGURE 3. Briefly, the CD20 antibody with a free sulfhydryl group attached to a lysine residue side chain in the Fc region is prepared by reacting the antibody with an affinity peptide configured to deliver a protected version of the sulfhydryl group (e.g., a thioester) to the lysine residue. The protecting group is then removed to reveal the free sulfhydryl. The free sulfhydryl is then reacted with a bifunctional reagent comprising a bromoacetamide group connected to the DBCO conjugation handle through a linking groupThe method can be used to produce an antibody with one DBCO group present (DARI) and / or two DBCO groups attached to the antibody (DAR2, one DBCO group linked to each Fc of the antibody). Unless otherwise specified, all immunocytokines described herein are DARI .WSGR Docket No. 56146-744.601

[0345] Conjugation of antibody to IL-18 polypeptide

[0346] The DBCO modified antibody is then conjugated to an IL-18 polypeptide comprising an azide moiety at a desired point of attachment (e.g., anIL-18 polypeptide which contains an amino acid with an azide side chain or an IL-18 linked to an azide using a bifunctional linking group as in Example 4). DBCO modified antibody with one (DARI) or two (DAR2) reactive handles are reacted with 2-10 equivalents of azide containing IL- 18 (pH 5.2 buffer, 5% trehalose, rt, 24 h). In an alternative embodiment, antibody comprising two DBCO conjugation handles is reacted either as an excess reagent (e.g., 5-10 equivalents) with 1 equivalent of IL- 18 comprising an azide functionality to produce a DARI antibody or the antibody comprising two DBCO conjugation handles is reacted with 1 equivalent of antibody with excess reagent of IL-18 comprising an azide (e.g., 5-10 equivalents) to produce a DAR2 antibody. An illustration of this protocol is shown inFIGURE 7

[0347] Purification and Characterization of antibody -IL- 18 immuno cytokine

[0348] The resulting immunocytokine is purified by cation -exchange chromatography and / or size exclusion chromatography to obtain purified immunocytokine. Antibody -IL- 18 polypeptide immunocytokine is purified from unreacted IL- 18 and aggregates using a desalting column, CIEX and SEC (GE Healthcare Life Sciences AKTA pure, mobile phase: Histidine 5.2 / 150 mM NaCl / 5% Trehalose, column: GE Healthcare Life Sciences SUPERDEX™ 200 increase 3.2 / 300, flow rate: 0.5 mL / min).

[0349] The purity and identity of the antibody-IL-18 polypeptide immunocytokine is confirmed by RP-HPLC (HPLC: ThermoFisher Scientific UHPLC Ultimate 3000, column: Waters BEH C- 4 300A, 3.0 pm, 4.6 mm, 250 mm, mobile phase A: 0.05% TFA in Water, mobile phaseB: 0.05% TFA in mixture of ACN:IPA:ETOH:H2O (5 : 1.5:2: 1.5), flow rate: 0.5 mL / min, injection amount: 10 pg (10 pL Injection of 1 mg / mL), gradient: 0% to 20% mobile phase B in 50 min) and SDS- PAGE.

[0350] Exemplary chromatograms and analytical characterization of DARI immunocytokines of an IL-18 of SEQ ID NO: 60 conjugated via residue C68 and an anti-PD-1 antibody (LZM-009) prepared according to the described methods are shown in FIGURES 8-10.

[0351] Additional exemplary chromatograms and analytical characterization of a DARI immunocytokine of an IL- 18 of SEQ ID NO: 30 conjugated via residue C68 to Fc residue K248 of the anti-PD-1 antibody LZM-009 are shown in FIGURE 11 (RP HPLC), FIGURE 12 (SEC- HPLC), and FIGURE 13 (Q-TOF Mass Spectrometry). The immunocytokine shown is Composition A. The linker is formed from bifunctional linking reagentWSGR Docket No. 56146-744.601attached to residue K248 of the antibody using AJIC APIMtechnology, then subsequently conjugated to the bromoacetamide -PEGs-azide linker attached to residue C68 of the IL-18 polypeptide.Example 4 - Characterization of Immunocytokine IL-18 Activity

[0352] The ability of the immunocytokine to perform various IL-18 activities is measured as provided below, as well as relevant comparisons to non -conjugated IL-18 polypeptides.Surface Plasmon Resonance

[0353] The interaction of immunocytokines, wild type IL-18, and of modified IL-18 polypeptides with human IL-18 receptor subunits are measured with Surface Plasmon Resonance (SPR) technology. Anti-human IgG antibodies are boundby amine coupling onto a CM5 chip to capture 6 pg / mL of Fc fused human IL-18Ra, 6 pg / mL of Fc fused human IL- 18RP, or 2 pg / mL of Fc fused human IL- 18BP isoform a (IL-18B Pa) for 30 min before capture. In other settings, 6 pg / mL of alpha and beta IL-18 receptors are mixed and pre-incubated for 30 min before capture of the alpha / beta heterodimer IL- 18 receptor.

[0354] The kinetic binding of the IL- 18 analytes and immunocytokines are measured with a Biacore 8K instrumentin two-fold serial dilutions startingat 1 pM down to 0.98 nM. Regeneration of the surface back to amine coupled anti IgG antibody is done after every concentration of analyte. To measure the protein association to the receptors, the samples are injected with a flow rate of 50 pL / min for 60 s, followed by 300 s buffer only to detect the dissociation. The used running buffer is IxPBS with 0.05% Tween20. The relative response units (RU, Y-axis) are plotted against time (s, X-axis) and analyzed in a kinetic 1 :1 binding model for the monomer receptor binding and for the binding to the IL-18BP. A kinetic heterogenous ligand fit model is applied for the alpha / beta heterodimer binding.IL-18BP Binding AlphaLISA® Assay

[0355] A human IL-18BP AlphaLISA® Assay Kit is used to determine the binding affinity of each immunocytokine and IL-18 variant for IL- 18BP, which detected the presence of free form IL-18BP.

[0356] Sixteen three-fold serial dilutions of IL-18 analytes are prepared in aMEM medium supplemented with 20% FCS, Glutamax, and 25 pM p -mercaptoethanol in the presence of 5 ng / mL of His-tagged human IL-18BP. Final IL-18 analytes concentration range from 2778 nM to 0.2 pM.WSGR Docket No. 56146-744.601

[0357] After 1 hr incubation at room temperature, free IL-18BP levels are measured using a Human IFNy AlphaLISA® Assay Kit. In a 384 well OPTIplate, 5 pL of 5X Anti -IL-18BP acceptor beads are added to 7.5 pL of an IL-18 / IL-18BP mix. After 30 min incubation at room temperature with shaking, 5 pL of biotinylated Anti-IL-18BP antibodies are added to each well. The plate is incubated further for 1 hr at room temperature. Under subdued light, 12.5 pL of 2X streptavidin (SA) donor beads are pipetted into each well, and the wells are incubated with shaking for an additional 30 min at room temperature. The AlphaLISA® signal is then measured on an Enspire plate reader with 680 and 615 nm as excitation and emission wavelengths, respectively. The dissociation constant (KD) is calculated based on a variable slope, four parameter analysis using GraphPad PRISM software.IFNy Induction Cellular Assay

[0358] The ability of immunocytokines and IL-18 polypeptides provided herein are assessed for ability to induce IFNy in a cellular assay according to the protocol below.

[0359] The NK cell line NK-92 derived from a patient with lymphoma (ATCC® CRL-2407™) is cultured in aMEM medium supplemented with 20% FCS, Glutamax, 25 pMB -mercaptoethanol, and 100 lU / mL of recombinant human IL-2.

[0360] On the day of experiment, cells are harvested and washed with aMEM medium without IL-2 and containing 1 ng / mL of recombinant human IL-12. After counting, cells are seeded at 100,000 cells / well in a 384 well titer plate and incubated at 37 °C / 5% CO2. Sixteen 4-fold serial dilutions of IL- 18 analytes are prepared in aMEM medium, and 1 ng / mL of IL- 12 were added to the NK-92 cells. Final IL-18 analyte concentrations range from 56 nM to 5xl0'5pM.

[0361] After incubating the cells for 16-20 hr at 37 °C / 5% CO2, 5 pL of supernatant is carefully transferred to a 384 microwell OptiPlate. IFNy levels are measured using a human IFNy AlphaLISA® Assay Kit. Briefly, 10 pL of 2.5X AlphaLISA® Anti-IFNy acceptor beads and biotinylated antibody anti-IFNy mix are added to the 5pL of NK-92 supernatants. The mixtures are incubated for 1 hr at room temperature with shaking. Under subdued light, 2.5 pL of 2X streptavidin (SA) donor beads are pipetted into each well, and the wells are incubated for 30 min at room temperature with shaking. AlphaLISA® signals are then measured on an EnSpire™ plate reader using 680 nm and 615 nm as excitation and emission wavelengths, respectively. Half maximal effective concentrations (EC50) are calculated based on a variable slope and four parameter analysis using GraphPad PRISM software.IL-18 Binding Protein Inhibition Cellular AssayWSGR Docket No. 56146-744.601

[0362] The NK cell line NK-92 derived from a patient with lymphoma (ATCC® CRL-2407™) is cultured in aMEM medium supplemented with 20% FCS-Glutamax, 25 pMB-mercaptoethanol, and 100 lU / mL of recombinant human IL-2.

[0363] On the day of experiment, cells are harvested and washed with aMEM medium without IL-2 and containing 1 ng / mL of recombinant human IL-12. After counting, the cells are seeded at 100,000 cells / well in a 384 well titer plate and incubated at 37 °C / 5% CO2. Sixteen 2-fold serial dilutions of Fc-fused human IL-18 binding protein isoform a (IL-18BPa) are prepared in aMEM medium . 1 ng / mL of IL- 12 containing 2 nM of each IL- 18 polypeptide variant is added to the NK- 92 cells. The final IL-18 analyte concentration is 1 nM, and the final IL-18BPa concentration ranged from 566 nM to 17 pM.After incubating the cells for 16-20 hr at 37 °C / 5% CO2, 5 pL of the supernatant is carefully transferred to a 384 microwell OptiPlate. IFNy levels are measured using a human IFNy AlphaLISA® Assay Kit. Briefly, 10 pL of 2.5X AlphaLISA® anti-IFNy acceptor beads and biotinylated antibody anti-IFNy mix are added to 5 pL of NK-92 supernatants. The mixtures are incubated for 1 hr at room temperature with shaking. Under subdued light, 2.5 pL of2X SA donor beads are pipetted in each well and incubated for 30 min at room temperature with shaking AlphaLISA® signals are then measured on an EnSpire™ plate reader using 680 nm and 615 nm as excitation and emission wavelengths, respectively. Half maximal inhibitory concentrations (IC50) are calculated based on a variable slope andfour parameter analysis using Grap hPadPRISM software.IFNy Induction on primary human cells

[0364] Ability of IL- 18 variants to stimulate Human peripheral blood mononuclear cells(PBMCs) was assessed according to the following protocol.

[0365] Isolation of lymphocytes: Blood from Buffy Coats of healthy volunteers was diluted with equal volume of PBS and slowly poured on top of SepMate tube prefilled with 15mL Histopaque- 1077. Tubes were centrifuged for 10 minutes at 1200g, the top layer was collected and washed 3 times with PBS containing 2% of Fetal Bovine Serum. PBMCs were counted and cryopreserved as aliquots of 20 * 106cells.

[0366] Cryopreserved PBMCs were thawed and seeded at 150 000 cells / well in a 96w round bottom 96 well plate. PBMCs were stimulated with a gradient of human IL-18 variants ranging from 0.2 pg / mL to 3600 ng / mL. All stimulations were performed in the presence of hIL-12 (Ing / ml, Sino Biological, #CT011 -H08H) for 24hrs in RPMI containing 10% Fetal Bovine Serum.WSGR Docket No. 56146-744.601

[0367] Cytokine production after 24hr stimulation were measured using LEGENDplex™ beadbased cytokine assay (Biolegend #740930) according to manufacturer protocol. Half maximal effective concentrations (EC50) of IFNy released in culture supernatant were calculated based on a variable slope and four parameter analysis using GraphPad PRISM software.IFNy Induction on primary mouse cells

[0368] Ability of IL- 18 variants to stimulate murine splenocytes was assessed according to the following protocol.

[0369] Cryopreserved splenocytesisolatedfromBALB / c and C57BL6 mice were purchased from IQ Biosciences (Berkeley, CA, USA).

[0370] Cryopreserved splenocytes were thawed, treated with DNAsel, and seeded at 200 000 cells / well in a 96w round bottom 96 well plate. Splenocytes were stimulated with a gradient of human IL-18 variants ranging from 0.2 pg / mL to 3600 ng / mL. All stimulations were performed in the presence of mIL-12 (Ing / ml, Peprotech, cat # 210-12) for 24hrs in RPMI containing 10% Fetal Bovine Serum.

[0371] Cytokine production after 24hr stimulation were measured using LEGENDplex™ beadbased cytokine assay (Biolegend #740622) according to manufacturer protocol. Half maximal effective concentrations (EC50) of IFNy released in culture supernatant were calculated based on a variable slope and four parameter analysis using GraphPad PRISM software.Example 5 - Immune Cell Associated Antigen Binding ELISA Assay (Figure 14A, Figure 14B, Figure 15A, and Figure 15B)

[0372] The interaction of the unmodified antibodies and corresponding IL-18 immunocytokines with relevant immune cell associated antigen are measured by ELISA assay. For these studies, Corning high -binding half-area plates (Fisher Scientific, Reinach, Switzerland) are coated overnight at 4°C with 25 pl of unmodified antibodies corresponding IL-18 immunocytokines at 5 pg / ml in PBS. Plates are then washed four times with 100 pl of PBS-0.02% Tween20. Plate surfaces are blocked with 25 pl of PBS-0.02% Tween20-l% BSA at 37°C during Ih. Plates are then washed fourtimes with 100 pl of PBS-0.02% Tween20. Twenty -five microliters (25 pl) of recombinant biotinylated human PD-1 (Biotinylated Recombinant Human PD-1 / CD279-Fc Chimera, carrier-free, Biolegend #789406) or PD-L1 (Biotinylated Human PD-L1 / B7-H1, ACROBiosy stems, PD1 -H82E5-25UG) protein are added in seven-fold serial dilutions starting at 12 nM down to 0.15 pM into PBS-0.02% Tween20-0.1%BSA and incubated at 37°C during 2h. Plates are then washed fourtimes with 100 pl of PBS-0.02% Tween20. Twenty -five microliters of Streptavidin- Horseradish peroxidase (#RABHRP3, Merck, Buchs, Switzerland) diluted at 1 :500 into PBS-0.02% Tween20-0.1% BSA are added to each well and incubated at RoomWSGR Docket No. 56146-744.601Temperature during 30 min. Plates are then washed four times with 100 pl of PBS-0.02% Tween20. Fifty microliters of TMB substrate reagent (#CL07, Merck, Buchs, Switzerland) are added to each well and incubated at 37°C during 5min. After 5 min at 37°C, Horseradish peroxidase reaction is stopped by adding 50 pl / well of 0.5MH2SO4stop solution. ELISA signal is then measured at 450 nm on an ENSPIRE® plate reader from Perkin Elmer (Schwerzenbach, Switzerland). Results from this experiment are shown in the table below.Table 4 - KD values of the interaction of immuno cytokines with PD-1 and PD-L1 as measured by ELISA

[0373] FIGURE 14A and FIGURE 14B show plots measuring ability of the unmodified and of conjugated anti-PDl antibodies to bind with humanPDl / CD279 ligand, with the figure showing ELISA signal on the y-axis and dosage of the biotinylated PD-1 protein on the x-axis. The unconjugated reference antibodies are Pembrolizumab, LZM-009, Nivolumab, Atezolizumab, Durvalumab, and Avelumab. The conjugated antibodies tested in this figure are compositions A and composition B.

[0374] FIGURE 15A and FIGURE 15B show plots measuring ability of the unmodified and of conjugated antibodiesto bind with human PD-L1 / B7-H1 ligand, with the figure showing ELISA signal on the y-axis and dosage of the biotinylated PD-L1 protein on the x-axis. The unconjugated reference antibodies are Pembrolizumab, LZM-009, Nivolumab, Atezolizumab, Durvalumab, and Avelumab. The conjugated antibodies tested in this figure are compositions A and composition B.WSGR Docket No. 56146-744.601Example 6 - Kinetic Analysis of Binding of reference antibodies and Immuno cytokines to Immune Cell Associated Antigens (Figure 16)

[0375] Based on Bio-Layer Interferometry (BLI), Octet® BLI systems enable real-time, label- free analysis for the determination of kinetics and affinity of a ligands to its receptor. Here anti- human IgGFC Capture (AHC) sensors are loaded with the test items (ICs) . Sensors are first dipped into a kinetic buffer for baseline measurement, then into an analyte solution, here human PD1, to allow association and again into a buffer solution where the analyte is allowed to come off the ligand (dissociation). Several concentrations of analyte are run in parallel and enable the calculation of affinity parameters: Ka, Kd, KD.

[0376] Typically, first, the sensors are regenerated by 3 cycles of dipping into 10 mM glycine solution atpH=2 for 20 seconds, followed by 20 second kinetics buffer and a final 60 seconds in kinetics buffer to establish the initial signal (baseline). Second, the loading column will contain the ligand, here the unmodified PD-1 antibodies and of IL-18 polypeptide conjugated PD-1 antibody, at a fixed concentration determined in the loading scout experiment (20 ug / mL). Then another wash / baseline step allows non immobilized proteins to be washed away. The association column will contain the 2-fold dilution series of the analyte (His-tagged human PD 1 , R&D #8986- PD) including a no analyte control. The highest concentration should be ~10-fold the KD. The dissociation designates the sensors to return to previous baseline column with kinetics buffer. After acquisition, the data is analyzed with Data Analysis Studio software (Sartorius). Data sets are first preprocessed by subtracting references samples and aligning curves on the baseline step. Group fittingis then applied to the data series and kinetics parameters are calculated. Results from this experiment are shown in the table below.Table 5 - Binding kinetics of the interaction of reference antibodies and immunocytokines with PD-1 as measured by Bio-Layer Interferometry (BLI)WSGR Docket No. 56146-744.601

[0377] FIGURE 16 shows plots measuring ability of the unmodified and of conjugated antibodies to bind to human PD-L1 / B7-H1 ligand, with the figure showing netBioLayer interferometry shift in nanometer on the y-axis and time of incubation dosage of the biotinylated PD-L1 protein on the x-axis. The figure shows mean ELISA signal on the y -axis and dosage of the human Fc gamma receptors on the x-axis. The unconjugated reference antibodies are Pembrolizumab and LZM-009. The conjugated antibodies tested is Compositions A.Example 7 - PD-1 / PD-L1 Blockade Assay (Figure 17A and 17B)

[0378] For immunocytokine compositions which comprise PD-1 orPD-Ll antibodies or antigen binding fragments, the experiment outlined below is performed to assess the ability of the immunocytokines to interfere with the PD-1 / PD-L1 pathway. The assay is the PD-1 / PD-L1 Blockade Bioassay from Promega (Cat# J1250, Madison, WI, USA). PD-1 / PD-L1 Blockade Bioassay is a bioluminescent cell -based assay based on the co-culture of effector cells with target cells mimicking an immunological synap se. Jurkat T cells expressing human PD- 1 and a luciferase reporter driven by a NF AT response element (NF AT -RE) are activated by CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate Jurkat cells cognate TCRs. Concurrent interaction PD-1 / PD-L1 inhibits TCR signaling and represses NFAT- RE-mediated luminescence. Addition of either an anti-PD-1 or anti-PD-Ll antibody thatblocks the PD-1 / PD-L1 interaction releases the inhibitory signal, restoring TCR activation and resulting in a gain of signal of NFAT-RE luminescent reporter.

[0379] Briefly, PD-L1 aAPC / CHO-Kl Target cells were plated in white tissue culture 96- wells plates and cultured overnight at 37°C / 5% CO2. Test molecules were measured in four-fold serial dilutions starting at 1 pM down to 0.002 nM and pre-incubated on target cells for 10 min before the addition of freshly thawed PD-1 Jurkat effector cells. After 6h at 37°C / 5% CO2, activity NFAT-RE luminescent reporter was evaluated by the addition of Bio -Gio reagent and measured on an ENSPIRE® plate reader (i sec / well) from Perkin Elmer (Schwerzenbach, Switzerland). Results from this experiment are shown in the table below.Table 6 - Activity of unconjugated IL-18 variants and corresponding IL-18 immunocytokines in the PD-1 / PD-L1 blockade cellular assayWSGR Docket No. 56146-744.601

[0380] FIGURE 17 A and 17B shows plots measuring ability of the unmodified and of conjugated anti-PDl antibodies to interfere with PD1 / PDL1 pathway, with the figure showing normalized luminescence intensity of effector cells NFAT-Lucia reporter on the y-axis and dosage of the unmodified and of conjugated anti-PDl antibodies on the x-axis. The unconjugated reference antibodies are Pembrolizumab, LZM-009, Nivolumab, Atezolizumab, Durvalumab, and Avelumab. The conjugated antibodies tested in this figure are compositions A and composition B.Example 8 - Human FcyR Binding Assay (Figure 18A and Figure 18B)

[0381] The interaction of the unmodified and of conjugated antibodies with human Fc gamma receptors I (FcyRI / CD64), with human Fc gamma receptors Ila (FcyRIIa / CD32a), with inhibitory human Fc gamma receptors lib (FcyRIIb / CD32b), and with human Fc gamma receptors III FcyRIIIa / CD16 were measured by ELISA.

[0382] Briefly, Corning high -binding half-area plates (Fisher Scientific, Reinach, Switzerland) were coated overnight at 4°C with 25 pl of unmodified and of conjugated anti-PDl antibodies at 2.5 pg / ml in PBS. Plates were then washed four times with 100 pl of PBS-0.02% Tween20. Plates surfaces were blocked with 25 pl of PBS-0.02% Tween20-l %B SA at 37°C during 1 h. Plates were then washed four times with 100 pl of PBS-0.02% Tween20. Then twenty -five microliters of either recombinant Human Fc gamma RI / CD64 Protein (R&D systems, 1257-FC-050, CF), recombinant Human Fc gamma RIIA / CD32a(H167) Protein (R&D systems, 9595-CD-050, CF), recombinant Human Fc gamma RIIB / CD32b Avi-tag Protein (R&D systems, A VII 875-050, CF), or recombinant Human Fc gamma RIIIA / CD 16a Protein (R&D systems, 4325-FC-050; CF) wereWSGR Docket No. 56146-744.601 added in five-fold serial dilutions ranging from 1000 nMto 0.001 nMinto PBS-0.02% Tween20- 0.1% BSA and incubated at 37°C during 2h. Plates were then washed four times with 100 pl of PBS-0.02% Tween20. Twenty -five microliters of a 1 / 500 HRP-anti-His antibody in PBS - 0.02% Tween20- 0.1%BSA (R&D systems, anti-HIS-HRP Ab, #MAB050H) were added to each well and plates were incubated atRoom Temperature during Ih. Plates were then washed four times with 100 pl of PBS-0.02% Tween20. Fifty microliters of TMB substrate reagent (#CL07, Merck, Buchs, Switzerland) were added to each well and incubated at 37°C during 5min. After 5min at 37°C, Horseradish peroxidase reaction was stopped by adding 50 pl / well of 0.5M H2SO4 stop solution. ELISA signal was then measured at 450 nm on an EnSpire plate reader from Perkin Elmer (Schwerzenbach, Switzerland). Results from this experiment are shown in the table below.Table 7 - Binding affinity of reference antibodies and immunocytokines with human Fc gamma Receptors as measured by ELISANT: Not Tested

[0383] FIGURE 18A and FIGURE 18B show plots measuring ability of the unmodified and of conjugated antibodies to bind to human Fc gamma receptor I (CD64) on top panels, and to human Fc gamma receptor Illa (CD 16) on lower panels. The figure shows mean ELISA signal on the y -WSGR Docket No. 56146-744.601 axis and dosage of the human Fc gamma receptors on the x-axis. The unconjugated reference antibodies are LZM-009 and Atezolizumab. The conjugated antibodies tested are Compositions A and B.Example 9 - Human FcRn Binding Assay (Figure 19)

[0384] The interaction of the unmodified and of conjugated anti-PDl antibodies with the human neonatal Fc receptor (FcRn) at pH 6 was measured using the AlphaLISA® Human FcRn Binding Kit (AL3095C) from Perkin Elmer (Schwerzenbach, Switzerland). The AlphaLISA® detection of FcRn and IgG binding uses IgG coated AlphaLISA® acceptor beads to interact with biotinylated human FcRn captured on Streptavidin -coated donor beads. When reference IgG binds to FcRn, donor and acceptor beads come into proximity enabling the transfer of singlet oxygen that trigger a cascade of energy transfer reactions in the acceptor beads, resulting in a sharp peak of light emission at 615 nm. Addition of a free IgG antibodies into the AlphaLISA® mixture creates a competition for the binding of FcRn to the reference antibody resulting in a loss of signal.

[0385] Briefly, test molecules were measured in serial dilutions starting at 5uM down to 64 pM and incubated with AlphaLISA® reaction mixture consisting of 800 nM of recombinant biotinylated human FcRn, 40 pg / ml of human IgG conjugated Acceptor beads, and 40 pg / ml of Streptavidin coated Donor beads in pH 6 MES buffer. After 90min at 23°C in the dark, AlphaLISA® signal was measured on an EnSpire plate reader (Excitation at 680 nm, Emission at 615 nm) from Perkin Elmer (Schwerzenbach, Switzerland). Results from this experiment are shown in the table below.Table 8 - Binding affinity of reference antibodies and immunocytokines with the human neonatal Fc receptor as measured by AlphaLISA®WSGR Docket No. 56146-744.601

[0386] FIGURE 19 shows plots measuring ability of the unmodified and of conjugated antibodies to bind to human Fc neonatal receptor. The figure shows mean AlphaLISA® signal on the y-axis and dosage of the human Fc neonatal receptor (FcRn) on the x-axis. The unconjugated reference antibodies are LZM-009 and Atezolizumab. The conjugated antibodies tested are Compositions A and B.Example 10 - IFN gamma secretion assay in NK92 cells (Figure 20 & 21)

[0387] The IFNy-secretion stimulating activity of the unconjugated IL-18 variants and corresponding IL-18 immunocytokines was evaluated on NK92 cell line. The NK cell line NK- 92 derived from a patient with lymphoma (ATCC, Cat# CRL-2407) was cultured in aMEM medium supplemented with 12.5%FCS, 12.5% horse serum (HS), 5 OuMB -mercaptoethanol, and 2 ng / ml of recombinant Human Interleukin-2 (IL-2).

[0388] On the day of experiment, cells were harvested and washed with aMEM medium without IL-2 and resuspended in medium (w / o IL-2) containing 1 ng / ml of recombinant human Interleukin- 12 (SinoBiologicals, Cat# CT011 -H08H). After counting, cells were seeded at 100 000 cells / well in a 384 well titer plate and incubated at 37°C / 5%CO2. Sixteen 4 -fold serial dilutions of IL- 18 analytes were prepared in aMEM medium- 1 ng / ml IL- 12 and were added to the NK-92 cells. Final IL-18 analytes concentration ranged from to 200 nM down to 0.01 pM.

[0389] After 16-20h incubation at 37°C / 5%CO2, 5 pl of supernatant were carefully transferred to a 384 microwells OPTIPlate (Perkin Elmer; Cat# 6007270) and Interferon -gamma (IFNy) levels measured using the Human IFNy AlphaLISA® Assay Kit (Perkin Elmer, Cat# AL217C). Briefly, 1 Opl of 2.5X AlphaLISA® Anti-IFNy acceptor beads and biotinylated Antibody Anti -IFNy mix were added to the 5 l of NK-92 supernatants and incubated for Ih at room temperature under shaking. Under subdued light, 2.5pl of 2X streptavidin (SA) donor beads were pipetted in each well and incubated for 30min at room temperature under shaking. AlphaLISA® signal was then measured on an Enspire plate reader (Perkin Elmer) using 680 and 615 nm as excitation and emission wavelengths respectively. Half maximal effective concentration (EC50) was calculated based on a variable slope, four parameter analysis using GraphPad PRISM software. Results from this experiment are shown in the table below.Table 9 - Activity of unconjugated IL-18 variants and corresponding IL-18 immunocytokines in the IFNy secretion NK92 assayWSGR Docket No. 56146-744.601NK92 cells

[0391] FIGURE 21 shows plots measuring ability of the unconjugated IL-18 variants and corresponding IL- 18 immunocytokines to stimulate the secretion of IFNy by NK92 cells. The figure shows mean IFNy AlphaLISA® signal on the y-axis and dosage of the unconjugated IL-18 variants and corresponding IL-18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL- 18 wild-type (SEQ ID N°:l), SEQ ID N°: 30, and SEQ ID N°: 31. Corresponding IL-18 immunocytokines tested are Compositions A, B, C, and D.Example 11 - IFN gamma secretion assay in KG-1 cells (Figure 22-24)

[0392] The IL-18 responsive AML cell line KG-1 shows high expression of IL-18Ra and moderate levels of IL18RP, respectively. The KG-1 cell line was used to generate a PD-1 expressing cell line and furthermore, to measure IFNy release upon incubation with IL- 18 variants and corresponding IL- 18 immunocytokines.

[0393] PD-1 expressing cell line generation: Briefly, KG-1 cells were transduced using lentiviral particles carrying the human PD-1 gene (PDCDl NM_005018; Origene, CAT#: RC210364L3V) at a MOI (Multiplicity of Infection) of 30. Spinfection was performed at 1260g during 90 min at 37°C in the presence of 5 pg / ml of Polybrene and 10 mM of HEPES in complete culture media (RPMI, 10% FBS, 1% L-Glutamine). Five days after transduction, puromycin at a final concentration of 1 ug / ml was added to select for PD-1 positive cells. For culture maintenance,WSGR Docket No. 56146-744.601 puromycin concentration was decreased to 0.5 pg / ml. Stable and homogenous expression ofPD- 1 was verified by surface staining (BD Pharmingen, #557860).

[0394] IFNy release was assessed in PD-1 positive (transduced) KG-1 cells, as well as in the parental PD-1 negative cells. 0.5xl05cells were seeded into a 96-well U-bottom plate in culture media (RPMI, 10% FBS, 1% L-Glutamine) and stimulated with IL-18 variants / ICs for 20-24h. The test items were diluted to lOOnM in culture medium, followed by 7 10 -fold serial dilutions. The lowest concentration assessed was 0.05 fM. After incubation, IFNy release was measured using the LEGENDplex™ custom human mix and match KIT (Biolegend LEGENDplex™ Human IFN-y Capture BeadB5, 13X#740942, LEGENDplex™ HU Essential Immune Response Panel Detection Abs, # 740931, LEGENDplex™ Buffer Set A # 740368). To this end, cell culture supernatant was collected and diluted 1 :1 with Assay Buffer. Fluorescence measurements were done with a QuanteonFlow Cytometer from Acea Biosciences. For analysis, MFI values (median fluorescence intensity) were exported and plotted against concentrations used. The EC 50 values (half maximal effective concentration) were calculated based on a variable slope and four parameter analysis using GraphPad PRISM software version 9. Results from this experiment are shown in the table below.Table 10 - Stimulation of IFNy secretion by unconjugated IL-18 variants and corresponding IL-18 immunocytokines in parental and PD-1 transduced KG-1 cellsWSGR Docket No. 56146-744.601KG-1 cells.

[0396] FIGURE 23 shows plots measuring the ability of the unconjugated IL-18 variants and corresponding IL-18 immunocytokines to stimulate the secretion of IFNyby parental PD-lne§atlveand by engineered PD-lP°sltlveKG-l cells. The figure shows mean IFNy LEGENDplex™ signal on the y-axis and dosage of the unconjugated IL-18 variants and corresponding IL-18 immunocytokines on the x-axis. The unconjugated IL- 18 variants are nativeIL-18 wild-type (SEQ ID N°:l), SEQ ID N°: 30, and SEQ ID N°: 31. Corresponding IL- 18 immunocytokines tested are Compositions A, B, C, and D.Example 12 - IL-18 Binding protein AlphaLISA® assay (Figure 24)

[0397] Wild type or modified IL-18 polypeptides samples were diluted at 5.6 pM in a solution of 1 x AlphaLISA® Immunoassay Buffer provided in the AlphaLISA® IFNy Detection kit and were diluted applying 3-fold serial dilutions down to 1.7pM in 384 deep well plates. A solution of lOng / ml of human IL-18BP-His was prepared with lx AlphaLISA® Immunoassay Buffer. IL- 18 / IL-18BP complex formation was performed incubating 3 Opl of IL-18BP solution to IL-18 sample titrations for Ih at 20°C. IL-18BP standard was prediluted from stock solution supplied in AlphaLISA® IFNy Detection kit at 1 OOng / ml with lx AlphaLISA® Buffer and titration prepared from applying 2 -fold serial dilutions. The following solutions were prepared: a 50pg / ml solution of anti-IL-18BP AlphaLISA® Acceptor beads, a 5nM solution of biotinylated anti-IL18BP antibody and an 80pg / ml light-protected solution of Streptavidin Donor beads in lx AlphaLISA® Immunoassay Buffer. To detect unbound IL- 18BP in IL-18 / IL-18P complex samples, 5 pl of premixed Acceptor beads solution were transferred on top on 7.5pL of samples in 384-well Optiplates, followed by a short centrifugation step at 150g, and incubated for 30 minutes at20°C under shaking at 750rpm.

[0398] 5 pl of Biotinylated anti-IL-18BP antibody were added, followed by a short centrifugation step at 150g, and incubated f or 60 minutes at 20°C under shaking at 75 Orpm . Under sub dued light, 12.5 pl of pre-mixed Donor beads were added, followedby a short centrifugation step at 150g, and incubated for 30 minutes at 20°C under shaking at 750rpm with no light. AlphaLISA® signal was then measured on an Enspire plate reader (Perkin Elmer) using 680 and 615 nm as excitation and emission wavelengths respectively. Unbound IL-18BP concentration interpolated from theWSGR Docket No. 56146-744.601 standard signal-concentration curve using GraphPad Prism. Results from this experiment are shown in the table below.Table 11 - Binding affinity of reference antibodies and immunocytokines with the humanIL-18 Binding Protein as measured by AlphaLISA®

[0399] FIGURE 24 shows plots measuring the ability of the unconjugated IL-18 variants and corresponding IL-18 immunocytokines to bind to the human IL-18 Binding Protein (IL-18BP). The figure shows mean free IL-18BP AlphaLISA® signal on the y-axis and dosage of the unconjugated IL-18 variants and corresponding IL- 18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL-18 wild-type (SEQ ID N°: 1), SEQ ID N°: 30, and SEQ ID N°: 31. Corresponding IL-18 immunocytokines tested are Compositions A, B, C, and D.Example 13 - Cellular IL-18 Binding protein resistance assay (Figure 25)

[0400] The NK cell line NK-92 derived from a patient with lymphoma (ATCC, Cat# CRL-2407) was cultured in aMEM medium supplemented with 20%FCS-Glutamax, 25 pM B- mercaptoethanol, and 100 lU / ml of recombinant Human Interleukin-2 (IL-2). On the day of experiment, cells were harvested and washed with aMEM medium without IL-2. After counting cells were seeded at 100 000 cells / well in a 384 well titer plate and incubated at 37°C / 5%CO2. Sixteen 2-fold serial dilutions of Fc fused human IL-18 binding protein isoform a (IL-18BPa; R&D systems, Cat# 119 -BP) were prepared in aMEM medium- Ing / ml IL- 12 containing 2nM of each IL-18 variants and were added to the NK-92 cells. Final IL-18 analytes concentration was InM and final IL-18BPa concentrations ranged from to 566nM down to 17pM.

[0401] After 16-20h incubation at 37°C / 5%CO2, 5 pl of supernatant were carefully transferred to a 384 microwells OPTIplate (Perkin Elmer; Cat# 6007270) and Interferon -gamma (IFNy) levelsWSGR Docket No. 56146-744.601 measured using the Human IFNy AlphaLISA® Assay Kit (Perkin Elmer, Cat# AL217C). Briefly, 1 Opl of 2.5X AlphaLISA® Anti-IFNy acceptor beads and biotinylated Antibody Anti -IFNy mix were added to the 5 pl of NK-92 supernatants and incubated for Ih at room temperature under shaking. Under subdued light, 2.5pl of 2X streptavidin (SA) donor beads were pipetted in each well and incubated for 30min at room temperature under shaking. AlphaLISA® signal was then measured on an Enspire plate reader (Perkin Elmer) using 680 and 615 nm as excitation and emission wavelengths respectively. Half maximal inhibitory concentration (IC50) was calculated based on a variable slope, four parameter analysis using GraphPad PRISM software. Results from this experiment are shown in the table below.Table 12 - IL-18BP-mediated inhibition of IFNy secretion by NK92 cells stimulated with unconjugated IL-18 variants and corresponding IL-18 immuno cytokines

[0402] FIGURE 25 shows plots measuring the ability of the human IL-18 Binding Protein to inhibit the secretion of IFNy by NK92 cells stimulated with 2nM of unconjugated IL-18 variants and corresponding IL- 18 immunocytokines. The figure shows mean IFNy AlphaLISA® signal on the y-axis and dosage of the human IL-18 Binding Protein on the x-axis. The unconjugated IL-18 variants are native IL-18 wild-type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. Corresponding IL-18 immunocytokines tested are Compositions A, B, C, and D.Example 14 - In Vivo Antitumor Activity in MC38 colon carcinoma model (Figure 26-27)

[0403] An in vivo efficacy study was performed in mice. Naive, 6-8 weeks old, C57BL / 6-hPDl female mice (GemPharmatech Co, Ltd, Nanjing, China) were inoculated subcutaneously at the right upper flank with MC38 tumor cells (3 x 105) in 0.1 mL of PBS for tumor development. TheWSGR Docket No. 56146-744.601 animals were randomized (using an Excel-based randomization software performing stratified randomization based upon tumor volumes), and treatment started when the average tumor volume reached approximately 120 mm3. Animals treated with unmodified antibodies received two weekly 10 mL / kg bolus int...

Claims

WSGR Docket No. 56146-744.601CLAIMSWHAT IS CLAIMED IS:1 . A method of treating cancer in a human subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of an immunocytokine composition, wherein the immunocytokine composition comprises: an IL-18 polypeptide; an antibody or antigen binding fragment thereof specific for programmed cell death protein 1 (PD-1); and a linker connecting the IL-18 polypeptide to the antibody or antigen binding fragment thereof.

2. The method of claim 1, wherein the IL- 18 polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1 .

3. The method of claim 1 or 2, wherein the IL-18 polypeptide exhibits a reduced ability to be inhibited by IL-18 binding protein (IL-18 BP) compared to the IL-18 polypeptide of SEQ ID NO: 1.

4. The method of any one of claims 1 -3, wherein the IL- 18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 30.

5. The method of any one of claims 1 -4, wherein the linker is attached to the IL-18 polypeptide at residue C68.

6. The method of any one of claims 1 -5, wherein the antibody or antigen binding fragment thereof comprises an Fc region.

7. The method of any one of claims 1 -6, wherein the antibody or antigen binding fragment thereof is a full-length antibody comprising an Fc region.

8. The method of claim 6 or 7, wherein the linker is attached to the Fc region Lys 246, Lys 248, Lys 288, Lys 290, or Lys 317 of the Fc region (EU numbering).

9. The method of claim 8, wherein the linker is attached to the Fc region at Lys 248 (EU numbering).

10. The method of any one of claims 1 -9, wherein the antibody or antigen binding fragment thereof is LZM-009 (Lipustobart).

11. The method of any one of claims 1 -10, wherein the linker comprises poly(ethylene glycol).

12. The method of any one of claims 1 -11, wherein the linker has a structureWSGR Docket No. 56146-744.60113. The method of claim 12, wherein the linker has a structure14. The method of any one of claims 1 -13, wherein the immunocytokine composition comprises only a single IL-18 polypeptide attached to the antibody or antigen binding fragment thereof.

15. The method of any one of claims 1 -14, wherein the cancer is resistant to treatment with at least one anti-cancer agent.

16. The method of claim 15, wherein the subject has failed to respond to treatment with the at least one anti-cancer agent.

17. The method of any one of claims 1 -16, wherein the cancer is resistant to treatment with an immune checkpoint inhibitor.

18. The method of claim 17, wherein the subject has failed to respond to treatment with the immune checkpoint inhibitor.

19. The method of any one of claims 1 -16, wherein the cancer in the subject has progressed after an initial response to treatment with an immune checkpoint inhibitor.

20. The method of any one of claims 1 -19, wherein the cancer is a metastatic or refractory tumor.

21. The method of any one of claims 1 -20, wherein the cancer is a locally advanced tumor.

22. The method of any one of claims 1 -21, wherein the cancer is a locally advanced unresectable tumor.

23. The method of any one of claims 1 -22, wherein the cancer is adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladderWSGR Docket No. 56146-744.601 cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pediatric cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cord cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureteral cancer, uterine cancer, vaginal cancer, or vulvar cancer.

24. The method of any one of claims 1 -22, wherein the cancer is non-small cell lung carcinoma (NSCLC), colorectal carcinoma (CRC), or squamous cell carcinoma of the head and neck (SCCHN).

25. The method of claim 24, wherein the cancer is NSCLC.

26. The method of claims 25, wherein the subject has previously received at least one of a platinum-based chemotherapy, a checkpoint inhibitor therapy, docetaxel, pemetrexed, or any combination thereof.

27. The method of claim 25, wherein the subject has previously received the checkpoint inhibitor therapy.

28. The method of claim 25, wherein the checkpoint inhibitor therapy was an anti-PD-1 or anti-PD-Ll antibody therapy.

29. The method of claim any one of claims 26-28, wherein the cancer is resistant to the prior treatment with the platinum-based chemotherapy, the checkpoint inhibitor therapy, docetaxel and / or pemetrexed.

30. The method of any one of claims 25-29, wherein the NSCLC lacks a sensitizing mutation.

31. The method of claim 30, wherein the NSCLCL lacks a sensitizing mutation in EGFR, ALK, ROS1, BRAF, MET (e.g., METexl4 skipping), RET, ERBB2, NTRK, or any combination thereof.

32. The method of claim 24, wherein the cancer is CRC.

33. The method of claim 32, wherein the CRC is micro satellite stable and / or proficient mismatch repair.

34. The method of claim 32 or 33, wherein the CRC is recurrent or intolerant after one or more prior regimen of chemotherapy.

35. The method of claim 34, wherein the prior regimen of chemotherapy comprises one or more of a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), oxaliplatin, irinotecan, an EGFR inhibitor (e.g., cetuximab, panitumumab, etc.), a VEGF / VEGFR2 pathway inhibitor (e.g., bevacizumab, ziv-aflibercept, ramucirumab, etc.), a thymidineWSGR Docket No. 56146-744.601 phosphorylase inhibitor / fluorpyrimidine combination therapy (e.g., tipiricil / trifluridine), an anti-HER2 agent, a multikinase inhibitor (e.g., regorafenib, fruquintinib, etc.), a BRAF inhibitor (e.g., encorafinib), an immune checkpoint inhibitor, or any combination thereof.

36. The method of any one of claims 32-35, wherein the CRC is KRAS, NRAS, and / or BRAF wild type CRC.

37. The method of claim 24, wherein the cancer is SCCHN.

38. The method of claim 37, wherein the subject has not received an immune checkpoint inhibitor therapy.

39. The method of claim 37, wherein the subject has previously received a platinum -based chemotherapy (e.g., cisplatin, carboplatin, etc.), the checkpoint inhibitor therapy, a fluoropyrimidine (e.g., 5 -fluorouracil, capecitabine, etc.), or any combination thereof.

40. The method of any one of claims 1 -39, wherein the immunocytokine composition is administered at a dose of from about 1 microgram / kg to about 1000 microgram / kg.

41. The method of any one of claims 1 -40, wherein the immunocytokine composition is administered at a dose of about 3 microgram / kg, about 10 microgram / kg, about 30 microgram / kg, about 100 microgram / kg, about 200 microgram / kg, about 300 microgram / kg, about 450 microgram / kg, or about 600 microgram / kg.

42. The method of any one of claim 1 -41, wherein the method comprises administering multiple doses of the immunocytokine composition to the subject.

43. The method of claim 42, wherein the multiple doses comprises 2, 3, 4, 5, or 6 doses.

44. The method of claim 42 or 43, wherein the multiple doses are administered about 2 weeks apart.

45. An aqueous pharmaceutical composition, comprising: a) an immunocytokine composition, comprising: an IL-18 polypeptide; and an antibody or an antigen binding fragment thereof specific for programmed cell death protein 1 (PD-1); and a linker, wherein the linker comprises: a first point of attachment to the IL-18 polypeptide; and a second point of attachment to the antibody or antigen binding fragment thereof; and b) a buffer, a carbohydrate, and a surfactant.WSGR Docket No. 56146-744.60146. The pharmaceutical composition of claim 45, wherein the buffer has a pKa value of about 6.

47. The pharmaceutical composition of claim 45 or 46, wherein the buffer comprises histidine / histidine hydrochloride.

48. The pharmaceutical composition of any one of claims 45-46, wherein the buffer is present in an amount of about 10 mM.

49. The pharmaceutical composition of any one of claims 45-48, wherein the carbohydrate comprises a sugar.

50. The pharmaceutical composition of claim 49, wherein the sugar is sucrose.

51. The pharmaceutical composition of any one of claims 45-50, wherein the carbohydrate is present in an amount of about 280 mM.

52. The pharmaceutical composition of any one of claims 45-51, wherein the surfactant is a polysorbate.

53. The pharmaceutical composition of claim 52, wherein the surfactant is polysorbate 80.

54. The pharmaceutical composition of any one of claims 45-53, wherein the surfactant is present in an amount of about 0.06 % (w / v).

55. The pharmaceutical composition of any one of claims 45-54, wherein pharmaceutical composition has a pH of about 6.2.

56. The pharmaceutical composition of any one of claims 45-55, wherein the pharmaceutical composition is stored as a frozen liquid.

57. The pharmaceutical composition of any one of claims 45-56, wherein the immunocytokine composition is Composition A.

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