TW202245843A - Skin cancer combination therapy with il-2 conjugates and cemiplimab - Google Patents

Abstract

Disclosed herein are methods for treating skin cancer in a subject in need thereof, comprising administering IL-2 conjugates in combination with cemiplimab.

Description

Combination therapy of IL-2 conjugate and cemiprimab (CEMIPLIMAB) for skin cancer

Disclosed herein are methods for treating skin cancer in a subject in need thereof comprising administering an IL-2 conjugate in combination with cimiprizumab.

Different T cell populations regulate the immune system to maintain immune homeostasis and tolerance. For example, regulatory T (Treg) cells prevent inappropriate responses of the immune system by preventing pathological autoreactivity, while cytotoxic T cells target and destroy infected cells and/or cancer cells. In some cases, modulation of distinct T cell populations provides options for treatment of a disease or indication.

Cytokines include a family of cell signaling proteins such as chemoattractants, interferons, interleukins, lymphokines, tumor necrosis factor, and other growth factors that play a role in innate and adaptive immune cell homeostasis. Cytokines are produced by immune cells such as macrophages, B-lymphocytes, T-lymphocytes and mast cells, endothelial cells, fibroblasts and different stromal cells. In some instances, cytokines regulate the balance between humoral and cell-based immune responses.

Interleukins are signaling proteins that regulate the development and differentiation of: T and B lymphocytes, cells of the monocyte lineage, neutrophils, basophils, eosinophils, megakaryocytes and hematopoietic cells. Interleukins are produced by helper CD4+ T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue-resident cells.

In some instances, interleukin 2 (IL-2) signaling is used to regulate T cell responses and subsequently to treat cancer. Accordingly, in one aspect, provided herein are methods of treating cancer in a subject comprising administering an IL-2 conjugate in combination with the anti-PD-1 antibody cimiprizumab.

Described herein is a method of treating skin cancer in a subject in need thereof comprising administering to the subject (a) an IL-2 conjugate and (b) cimiprizumab, wherein the IL -2 The conjugate comprises the amino acid sequence of SEQ ID NO: 1 having an unnatural amino acid residue described herein at position 64, such as the amino acid sequence of SEQ ID NO: 2.

In some embodiments, administration of the IL-2 conjugate and cimiprizumab provides improved outcomes in the treatment of skin cancer or a subtype thereof relative to existing therapies. For example, improved outcomes may be in terms of frequency of favorable outcomes, such as complete response, elimination of target lesions, reduction in size of target lesions, partial responses, stable disease, or slowed growth of target lesions. In some embodiments, the IL-2 conjugate and simiprizumab are administered relative to an existing skin cancer or IL-2 therapy, or relative to monotherapy using the IL-2 conjugate or simiprizumab alone. Mipilimumab offered an improved safety profile. For example, improved safety may be in terms of avoiding or reducing the frequency of adverse events, such as grade 4 adverse events; vascular leak syndrome (eg, grade 2, 3 and/or 4 vascular leak syndrome); Capillary leak syndrome; extravasation of plasma proteins and fluids into the extravascular space of the subject; hypotension and/or decreased organ perfusion in the subject; impaired neutrophil function in the subject; A decrease in mean arterial blood pressure in the subject; a systolic blood pressure less than 90 mm Hg or a fall in systolic blood pressure of 20 mm Hg from baseline; eosinophilia; edema or impaired renal or hepatic function; or decreased chemotaxis in the subject . In some embodiments, improved safety may be in that: the subject is not at increased risk of disseminated infection; the subject is not exacerbating pre-existing or initial manifestations of an autoimmune or inflammatory disorder. In some embodiments, administering the IL-2 conjugate and cimiprizumab provides improved results in that one or more of the favorable outcomes discussed above or disclosed elsewhere herein or at a frequency comparable to the above A combination of one or more of the safety improvements discussed or disclosed elsewhere herein.

Exemplary embodiments include the following.

式 (I) 其中： Z是CH ₂並且Y是

； Y是CH ₂並且Z是

； Z是CH ₂並且Y是

；或者 Y是CH ₂並且Z是

； W是具有約25 kDa-35 kDa的平均分子量的PEG基團； q是1、2或3； X是具有以下結構的L-胺基酸：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與後一個胺基酸殘基的附接點。 Embodiment 1. A method of treating skin cancer in a subject in need thereof comprising administering to the subject (a) an IL-2 conjugate, and (b) cimiprizumab, wherein : The IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is replaced by a structure of formula (I):

Formula (I) where: Z is _CH and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid with the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue.

式 (I) 其中： Z是CH ₂並且Y是

； Y是CH ₂並且Z是

； Z是CH ₂並且Y是

；或者 Y是CH ₂並且Z是

； W是具有約25 kDa-35 kDa的平均分子量的PEG基團； q是1、2或3； X是具有以下結構的L-胺基酸：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與後一個胺基酸殘基的附接點。 Embodiment 2. A method of treating skin cancer in a subject in need thereof, comprising administering to said subject (a) an IL-2 conjugate, and (b) cimiprizumab, wherein : the skin cancer is unresectable skin cancer, locally advanced cutaneous squamous cell carcinoma, or metastatic skin cancer; and the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein at position P64 Amino acids are replaced by structures of formula (I):

Formula (I) where: Z is _CH and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid with the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue.

式 (I) 其中： Z是CH ₂並且Y是

； Y是CH ₂並且Z是

； Z是CH ₂並且Y是

；或者 Y是CH ₂並且Z是

； W是具有約25 kDa-35 kDa的平均分子量的PEG基團； q是1、2或3； X是具有以下結構的L-胺基酸：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與後一個胺基酸殘基的附接點。 Embodiment 3. A method of treating skin cancer in a subject in need thereof, comprising: selecting a subject with skin cancer, wherein the subject is selected based on one or more attributes, the attributes comprising ( i) the skin cancer is unresectable skin cancer; (ii) the skin cancer is locally advanced cutaneous squamous cell carcinoma; or (iii) the skin cancer is metastatic skin cancer; and to the subject administering (a) an IL-2 conjugate, and (b) cimiprizumab, wherein: the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amine group at position P64 The acid is replaced by a structure of formula (I):

Formula (I) where: Z is _CH and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid having the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue.

式 (I) 其中： Z是CH ₂並且Y是

； Y是CH ₂並且Z是

； Z是CH ₂並且Y是

；或者 Y是CH ₂並且Z是

； W是具有約25 kDa-35 kDa的平均分子量的PEG基團； q是1、2或3； X是具有以下結構的L-胺基酸：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與後一個胺基酸殘基的附接點。 Embodiment 4. A method of treating skin cancer in a subject in need thereof comprising administering to said subject (a) about 8 μg/kg, 16 μg/kg or 24 μg/kg of IL- 2 conjugates, and (b) simiprizumab, wherein: the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is represented by formula (I) Structural substitution:

Formula (I) where: Z is _CH and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid having the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue.

Embodiment 5. The method of any one of embodiments 1-4, wherein the skin cancer is melanoma.

Embodiment 6. The method of any one of embodiments 1-4, wherein the skin cancer is squamous cell carcinoma of the skin.

Embodiment 7. The method of any one of embodiments 1-4, wherein the skin cancer is locally advanced squamous cell carcinoma of the skin.

Embodiment 8. The method of any one of embodiments 1-7, wherein the skin cancer is unresectable.

Embodiment 9. The method of any one of embodiments 1-8, wherein the skin cancer is metastatic.

Embodiment 10. The method of any one of embodiments 1-9, wherein the skin cancer is not amenable to topical therapy.

Embodiment 11. The method of any one of embodiments 1-10, wherein the skin cancer is advanced.

Embodiment 12. The method of any one of embodiments 1-11, wherein the skin cancer is immune checkpoint inhibitor-naïve.

Embodiment 13. The method of any one of embodiments 1-12, comprising administering to the subject about 8 μg/kg of the IL-2 conjugate.

Embodiment 14. The method of any one of embodiments 1-12, comprising administering to the subject about 16 μg/kg of the IL-2 conjugate.

Embodiment 15. The method of any one of embodiments 1-12, comprising administering to the subject about 24 μg/kg of the IL-2 conjugate.

Embodiment 16. The method of any one of embodiments 1-15, wherein in the IL-2 conjugate, the PEG group has an average molecular weight of about 30 kDa.

。 Embodiment 17. The method of any one of embodiments 1-16, wherein in the IL-2 conjugate, Z is _CH and Y is

.

。 Embodiment 18. The method of any one of embodiments 1-16, wherein in the IL-2 conjugate, Y is _CH and Z is

.

。 Embodiment 19. The method of any one of embodiments 1-16, wherein in the IL-2 conjugate, Z is _CH and Y is

.

。 Embodiment 20. The method of any one of embodiments 1-16, wherein in the IL-2 conjugate, Y is _CH and Z is

.

式 (IV)；

式 (V)； 其中： W是具有約25 kDa-35 kDa的平均分子量的PEG基團； q是1、2或3； X是具有以下結構的L-胺基酸：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與後一個胺基酸殘基的附接點。 Embodiment 21. The method of any one of embodiments 1-16, wherein the structure of formula (I) has the structure of formula (IV) or formula (V), or is of formula (IV) and formula (V) mixture:

Formula (IV);

Formula (V); wherein: W is a PEG group having an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid having the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue.

式 (XII)；

式 (XIII)； 其中： n是整數，使得-(OCH ₂CH ₂) _n-OCH ₃具有約30 kDa的分子量； q是1、2或3；以及 波浪線表示與SEQ ID NO: 1中未被替代的胺基酸殘基的共價鍵。 Embodiment 22. The method of any one of embodiments 1-16, wherein the structure of formula (I) has the structure of formula (XII) or formula (XIII), or is of formula (XII) and formula (XIII) mixture:

Formula (XII);

Formula (XIII); wherein: n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 30 kDa; q is 1, 2 or 3; A covalent bond to the amino acid residue being substituted.

Embodiment 23. The method of any one of embodiments 1-22, wherein q is 1.

Embodiment 24. The method of any one of Embodiments 1-23, wherein q is 2.

Embodiment 25. The method of any one of Embodiments 1-24, wherein q is 3.

Embodiment 26. The method of any one of embodiments 1-25, wherein the IL-2 is administered to the subject about once every two weeks, about once every three weeks, or about once every four weeks Joints.

Embodiment 27. The method of any one of embodiments 1-26, wherein the IL-2 is administered to the subject about once every two weeks, about once every three weeks, or about once every four weeks Conjugates and simiprizumab.

Embodiment 28. The method of any one of embodiments 1-27, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate or hydrate.

Embodiment 29. The method of any one of embodiments 1-28, wherein cimiprizumab is administered every 3 weeks at a dose of about 350 mg.

Embodiment 30. The method of any one of embodiments 1-29, wherein the IL-2 conjugate and cimiprizumab are administered separately.

Embodiment 31. The method of embodiment 30, wherein the IL-2 conjugate and cimiprizumab are administered sequentially.

Embodiment 32. The method of embodiment 30 or 31, wherein the IL-2 conjugate is administered prior to cimiprizumab.

Embodiment 33. The method of embodiment 30 or 31, wherein the IL-2 conjugate is administered after simiprizumab.

Embodiment 34. The method of any one of embodiments 1-33, wherein the IL-2 conjugate is administered to the subject by intravenous administration.

Embodiment 35. The method of any one of embodiments 1-34, wherein the IL-2 conjugate and cimiprizumab are administered to the subject by intravenous administration.

Embodiment 36. The method of any one of embodiments 1-35, further comprising administering acetaminophen to the subject.

Embodiment 37. The method of any one of embodiments 1-36, further comprising administering diphenhydramine to the subject.

Embodiment 38. The method of any one of embodiments 1-37, further comprising administering to the subject ondansetron.

Embodiment 39. The method of any one of embodiments 36-38, wherein acetaminophen, diphenhydramine, and/or ondansetron are administered to the subject prior to administration of the IL-2 conjugate. tester.

Embodiment 40. The method of any one of embodiments 1-39, further comprising selectively administering the IL-2 conjugate and simetrop based at least in part on the skin cancer being unresectable skin cancer Limumab subjects.

Embodiment 41. The method of any one of embodiments 1-40, further comprising selectively administering the IL-2 conjugate and Western cutaneous squamous cell carcinoma based at least in part on the skin cancer being locally advanced squamous cell carcinoma of the skin. Mipilimumab subjects.

Embodiment 42. The method of any one of embodiments 1-41, further comprising selectively administering the IL-2 conjugate and cimipril based at least in part on the skin cancer being metastatic skin cancer Monoclonal antibody subjects.

Embodiment 43. The method of any one of embodiments 1-42, further comprising selecting to administer the IL-2 conjugate and cimipril mono based at least in part on the skin cancer being unsuitable for topical therapy. resistant subjects.

Embodiment 44. The method of any one of embodiments 1-43, further comprising selecting to administer the IL-2 conjugate based at least in part on the skin cancer being immune checkpoint inhibitor naïve and simiprizumab subjects.

Embodiment 45. An IL-2 conjugate for use in the method of any one of embodiments 1-44.

Example 46. Use of an IL-2 conjugate in the manufacture of a medicament for use in the method of any one of examples 1-45.

Cross References to Related Applications

This application claims priority to U.S. Provisional Application No. 63/149,081, filed February 12, 2021, and U.S. Provisional Application No. 63/276,955, filed November 8, 2021, the disclosure of each of which is hereby incorporated by reference in its entirety is hereby incorporated. definition

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 claimed subject matter belongs. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter that is claimed. In the event that any material incorporated herein by reference is inconsistent with the express content of this disclosure, the express content controls. In this application, the use of the singular includes the plural unless expressly stated otherwise. It must be noted that, as used in the specification and appended claims, the singular forms "a, an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless the context requires otherwise. Furthermore, the use of the term "including" as well as other forms such as "include", "includes" and "included" is not limiting.

Reference in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some of the present disclosure. Examples, but not necessarily included in all embodiments of the present invention.

As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes exact amounts. Thus, "about 5 μL" means "about 5 μL" and also means "5 μL". In general, the term "about" includes an amount that is expected to be within experimental error, such as, for example, within 15%, 10%, or 5%.

The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

As used herein, the terms "subject" and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. None of the terms require or are limited to situations characterized by supervision (eg, continuous or intermittent) by a health care worker (eg, physician, registered nurse, nurse practitioner, physician assistant, paramedic, or hospice worker).

As used herein, the term "non-natural amino acid" refers to an amino acid other than one of the 20 naturally occurring amino acids. Exemplary unnatural amino acids are described in Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry 285 (15): 11039-11044 (2010), the disclosure of which is accessed by incorporated herein by reference.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single-chain antibody molecules (such as scFv); Sexual antibodies.

As used herein, "nucleotide" refers to a compound comprising a nucleoside moiety and a phosphate moiety. Exemplary natural nucleotides include, without limitation, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), adenosine diphosphate (ADP), Uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), and guanosine Monophosphate (GMP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine Diphosphate (dADP), thymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP), deoxyguanosine diphosphate (dGDP), deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate (dTMP), deoxycytidine monophosphate (dCMP) and deoxyguanosine monophosphate (dGMP). Exemplary natural deoxyribonucleotides comprising deoxyribose as the sugar moiety include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary natural ribonucleotides that contain ribose as the sugar moiety include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.

As used herein, "base" or "nucleobase" refers to at least the nucleobase portion of a nucleoside or nucleotide (nucleosides and nucleotides encompass ribose or deoxyribose variants), which or Nucleotides may in some cases contain further modifications to the nucleoside or sugar moiety of the nucleotide. In some contexts, "base" is also used to refer to an entire nucleoside or nucleotide (eg, a "base" may be incorporated into DNA by DNA polymerase, or into RNA by RNA polymerase). However, unless the context requires, the term "base" should not be interpreted as necessarily representing an entire nucleoside or nucleotide. In the base or nucleobase chemical structures provided herein, only the base of the nucleoside or nucleotide is shown, with the sugar moiety and optionally any phosphate residues omitted for clarity. As used in the chemical structures of bases or nucleobases provided herein, wavy lines represent linkages to nucleosides or nucleotides, where the sugar moiety of the nucleosides or nucleotides may be further modified. In some embodiments, the wavy line represents the attachment of a base or nucleobase to a nucleoside or sugar moiety of a nucleotide, such as a pentose sugar. In some embodiments, the pentose sugar is ribose or deoxyribose.

In some embodiments, the nucleobase is typically the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring, may be modified, may bear no similarity to natural bases, and/or may be synthetic, eg, by organic synthesis. In certain embodiments, a nucleobase comprises any atom or group of atoms in a nucleoside or nucleotide, wherein said atom or group of atoms is capable of bonding with a base of another nucleic acid with or without hydrogen bonding interaction. In certain embodiments, non-natural nucleobases are not derived from natural nucleobases. It should be noted that non-natural nucleobases do not necessarily have base properties, but for simplicity they are referred to as nucleobases. In some embodiments, when referring to a nucleobase, "(d)" indicates that the nucleobase may be attached to deoxyribose or ribose, while "d" without brackets indicates that the nucleobase is attached to deoxyribose .

As used herein, a "nucleoside" is a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA), abasic nucleosides, modified nucleosides, and nucleosides with mimic bases and/or sugar groups. Nucleosides include nucleosides containing substituents of any kind. A nucleoside may be a glycosidic compound formed by a glycosidic linkage between a nucleic acid base and a reducing group of a sugar.

As used herein, the term "analogue" of a chemical structure refers to a chemical structure that retains substantial similarity to the parent structure but which may not be readily synthesized from the parent structure. In some embodiments, nucleotide analogs are non-natural nucleotides. In some embodiments, nucleoside analogs are unnatural nucleosides. Related chemical structures that are readily synthesized from a parent chemical structure are termed "derivatives".

As used herein, a "dose-limiting toxicity" (DLT) is defined as an adverse event occurring within ± 1 day from Day 1 to Day 29 (inclusive) of a treatment cycle that is not clearly or undisputedly related only to Extrinsic causes are relevant and meet the criteria described for DLT in Example 2.

As used herein, "severe cytokine release syndrome" refers to grade 4 or grade 5 cytokine release syndrome as described in Teachey et al., Cancer Discov. 2016; 6(6); 664-79, the disclosure of which is incorporated by reference Incorporated into this article.

As used herein, "simiprizumab" refers to the human anti-PD-1 antibody sold under the trade name "Libtayo" by Regeneron Pharmaceuticals, Inc. and Sanofi-Aventis.

Although various features of the invention may be described in the context of a single embodiment, said features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment. IL-2 conjugate

Interleukin 2 (IL-2) is a pleiotropic type 1 cytokine whose structure consists of four α-helical bundles of 15.5 kDa. The precursor form of IL-2 is 153 amino acid residues in length, of which the first 20 amino acids form the signal peptide and residues 21-153 form the mature form. IL-2 is mainly produced by CD4+ T cells following antigenic stimulation, and to a lesser extent by CD8+ cells, natural killer (NK) cells, and natural killer T (NKT) cells, primed dendritic cells (DC), and mast cells produce. IL-2 signaling works by interacting with the IL-2 receptor (IL-2R) subunits, IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122) and IL-2Rγ (also known as CD132) specific combinations of interactions. Interaction of IL-2 with IL-2Rα forms a "low affinity" IL-2 receptor complex with a K _d of about 10 ^-8 M. Interaction of IL-2 with IL-2Rβ and IL-2Rγ forms a "medium affinity" IL-2 receptor complex with a K _d of about 10 ⁻⁹ M. Interaction of IL-2 with all three subunits IL-2Rα, IL-2Rβ and IL- _2Rγ forms a "high affinity" IL-2 receptor complex with a Kd of approximately >10 ⁻¹¹ M.

In some instances, IL-2 signaling through the "high affinity" IL-2Rαβγ complex regulates the initiation and proliferation of regulatory T cells. Regulatory T cells or CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T (Treg) cells mediate immune homeostasis by suppressing effector cells such as CD4 ⁺ T cells, CD8 ⁺ T cells, B cells, NK cells and NKT cells maintenance. In some cases, Treg cells were generated from the thymus (tTreg cells) or induced from peripheral naive T cells (pTreg cells). In some contexts, Treg cells are considered mediators of peripheral tolerance. Indeed, in one study, transfer of CD25-depleted peripheral CD4 ⁺ T cells produced multiple autoimmune diseases in nude mice, whereas co-transfer of CD4 ⁺ CD25 ⁺ T cells inhibited the development of autoimmunity (Sakaguchi, et al. , "Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25)," J. Immunol. 155(3): 1151-1164 (1995), the disclosure of which is incorporated herein by reference) . Increased Treg populations downregulate effector T cell proliferation and suppress autoimmunity and T cell antitumor responses.

IL-2 signaling via the "medium affinity" IL-2Rβγ complex regulates the initiation and proliferation of CD8 ⁺ effector T (Teff) cells, NK cells and NKT cells. CD8 ⁺ Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, Tcon or killer T cells) are cells that recognize and kill damaged cells, cancer cells and T lymphocytes of pathogen-infected cells. NK and NKT cells are lymphocyte types similar to CD8 ⁺ Teff cells that target cancer cells and pathogen-infected cells.

In some instances, IL-2 signaling is used to modulate T cell responses and subsequently to treat cancer. For example, IL-2 is administered in high doses to induce expansion of Teff cell populations for the treatment of cancer. However, high doses of IL-2 further lead to concomitant stimulation of Treg cells, thereby attenuating antitumor immune responses. High doses of IL-2 also induce toxic adverse events mediated by the engagement of IL-2Rα chain expressing cells in the vasculature, including type 2 innate immune cells (ILC-2), eosinophils, and endothelial cells. This leads to eosinophilia, capillary leak and vascular leak syndrome (VLS).

Adoptive cell therapy allows physicians to effectively use a patient's own immune cells to fight diseases, such as proliferative diseases (eg, cancer) and infectious diseases. The effects of IL-2 signaling can be further enhanced by the presence of additional agents or approaches in combination therapy. For example, programmed cell death protein 1, also known as PD-1 or CD279, is a cell surface receptor expressed on T cells and progenitor B cells that plays a role in regulating the immune system's response to the body's cells. PD-1 downregulates the immune system and promotes self-tolerance by inhibiting T cell inflammatory activity. This prevents autoimmune disease, but also prevents the immune system from killing cancer cells. PD-1 prevents autoimmunity through two mechanisms. First, PD-1 promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, PD-1 reduces apoptosis in regulatory T cells (anti-inflammatory suppressor T cells). Cimiprizumab, a human anti-PD-1 antibody that blocks PD-1 and primes the immune system to attack tumors, is approved to treat certain squamous cell skin cancers.

Provided herein are methods of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate, and (b) cimiprizumab.

(I) 其中： Z是CH ₂並且Y是

； Y是CH ₂並且Z是

； Z是CH ₂並且Y是

；或者 Y是CH ₂並且Z是

； W是具有約25 kDa-35 kDa的平均分子量的PEG基團； q是1、2或3； X是具有以下結構的L-胺基酸：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與後一個胺基酸殘基的附接點。 In some embodiments, the IL-2 sequence comprises the sequence of SEQ ID NO: 1: PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 1) wherein the amino acid at position P64 of the formula (

(I) where: Z is _CH2 and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid with the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue.

In any embodiment or variation of Formula (I) described herein, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate or hydrate. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt. In some embodiments, the IL-2 conjugate is a solvate. In some embodiments, the IL-2 conjugate is a hydrate.

In any embodiment or variation of formula (I) and pharmaceutical compositions comprising the same described herein, the average molecular weight encompasses both weight average molecular weight and number average molecular weight; in other words, for example, 30 kDa number average molecular weight and 30 kDa Both kDa weight average molecular weights correspond to 30 kDa molecular weights. In some embodiments, the average molecular weight is a weight average molecular weight. In other embodiments, the average molecular weight is a number average molecular weight. It is understood that in the methods provided herein, administering to a subject an IL-2 conjugate as described herein includes administering more than a single molecule of an IL-2 conjugate; therefore, the term "average" is used to describe PEG The molecular weight of the group refers to the average molecular weight of the PEG group of the IL-2 conjugate molecule in the dose administered to the subject.

。在式(I)的一些實施例中，Y是CH ₂並且Z是

。在式(I)的一些實施例中，Z是CH ₂並且Y是

。在式(I)的一些實施例中，Y是CH ₂並且Z是

。 In some embodiments of formula (I), Z is _CH and Y is

. In some embodiments of formula (I), Y is _CH and Z is

. In some embodiments of formula (I), Z is _CH and Y is

. In some embodiments of formula (I), Y is _CH and Z is

.

In some embodiments of formula (I), q is 1. In some embodiments of formula (I), q is 2. In some embodiments of formula (I), q is 3.

In some embodiments of formula (I), W is a PEG group having an average molecular weight of about 25 kDa. In some embodiments of formula (I), W is a PEG group having an average molecular weight of about 30 kDa. In some embodiments of formula (I), W is a PEG group having an average molecular weight of about 35 kDa.

(Ia) 其中： Z是CH ₂並且Y是

； Y是CH ₂並且Z是

； Z是CH ₂並且Y是

；或者 Y是CH ₂並且Z是

； W是具有約25 kDa-35 kDa的平均分子量的PEG基團； X是具有以下結構的L-胺基酸：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與後一個胺基酸殘基的附接點。 In some embodiments of formula (I), q is 1 and the structure of formula (I) is the structure of formula (Ia):

(Ia) where: Z is _CH2 and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; X is an L-amino acid with the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue.

。在式(Ia)的一些實施例中，Y是CH ₂並且Z是

。在式(Ia)的其他實施例中，Z是CH ₂並且Y是

。在式(Ia)的一些實施例中，Y是CH ₂並且Z是

。 In some embodiments of formula (Ia), Z is _CH and Y is

. In some embodiments of formula (Ia), Y is _CH and Z is

. In other embodiments of formula (Ia), Z is _CH and Y is

. In some embodiments of formula (Ia), Y is _CH and Z is

.

In some embodiments of Formula (Ia), the PEG group has an average molecular weight of about 30 kDa.

在一些實施例中，IL-2接合物包含SEQ ID NO: 2的序列： PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 2) 其中[AzK_L1_PEG30kD]是經由DBCO介導的點擊化學與PEG穩定接合的N6 -((2-azidoethoxy)-carbonyl)-L-lysine, the click chemistry forms a compound comprising a structure of formula (IV) or formula (V), wherein q is 1 (such as formula ( IVa) or formula (Va)), and wherein the PEG group has an average molecular weight of about 25-35 kilodaltons (eg, about 30 kDa), capped with a methoxy group. The term "DBCO" means a chemical moiety comprising a dibenzocyclooctynyl group, such as includes the mPEG-DBCO compounds shown in Schemes 1 and 2 of Example 1 .

The ratio of positional isomers generated from the click reaction is about 1:1 or greater than 1:1.

A PEG will generally contain many (OCH ₂ CH ₂ ) monomers (or (CH ₂ CH ₂ O) monomers, depending on how PEG is defined). In some embodiments, the amount of (OCH ₂ CH ₂ ) monomer (or (CH ₂ CH ₂ O) monomer) is such that the average molecular weight of the PEG group is about 30 kDa.

In some cases, PEG is a capped polymer, that is, a polymer whose at least one end is capped with a relatively inert group such as a lower C _1-6 alkoxy or hydroxyl group. In some embodiments, the PEG group is methoxy-PEG (commonly referred to as mPEG), which is a linear form of PEG in which one end of the polymer is a methoxy (-OCH ₃ ) group and the other Terminating in a hydroxyl group or other functional group which may optionally be chemically modified.

In some embodiments, the PEG group is a linear or branched PEG group. In some embodiments, the PEG group is a linear PEG group. In some embodiments, the PEG group is a branched PEG group. In some embodiments, the PEG group is a methoxy PEG group. In some embodiments, the PEG group is a linear or branched methoxy PEG group. In some embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. For example, IL-2 conjugates comprising a PEG group with a molecular weight of 30,000 Da ± 3,000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 5,000 Da are within the scope of this disclosure.

式 (IV)；

式 (V)； 其中： W是具有約25 kDa-35 kDa的平均分子量的PEG基團； q是1、2或3；以及 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與後一個胺基酸殘基的附接點。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein amino acid residue P64 is represented by formula (IV) or formula (V) or formula (IV) and formula ( V) Structural substitution of mixtures of:

Formula (IV);

Formula (V); wherein: W is a PEG group having an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; and X has the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue.

In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) or Formula (V), q is 1. In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) or Formula (V), q is 2. In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) or Formula (V), q is 3.

In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) or Formula (V), W is a PEG group having an average molecular weight of about 25 kDa. In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) or Formula (V), W is a PEG group having an average molecular weight of about 30 kDa. In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) or Formula (V), W is a PEG group having an average molecular weight of about 35 kDa.

In any of the embodiments described herein, the structure of Formula (I) has the structure of Formula (IV) or Formula (V), or is a mixture of Formula (IV) and Formula (V). In some embodiments, the structure of Formula (I) has the structure of Formula (IV). In some embodiments, the structure of Formula (I) has the structure of Formula (V). In some embodiments, the structure of Formula (I) is a mixture of Formula (IV) and Formula (V).

式 (IVa)；

式 (Va)； 其中： W是具有約25 kDa-35 kDa的平均分子量的PEG基團；以及 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與後一個胺基酸殘基的附接點。 In some embodiments of formula (IV) or formula (V), or a mixture of formula (IV) and formula (V), q is 1, the structure of formula (IV) is the structure of formula (IVa), and the formula ( The structure of V) is the structure of formula (Va):

Formula (IVa);

Formula (Va); wherein: W is a PEG group having an average molecular weight of about 25 kDa-35 kDa; and X has the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue.

In some embodiments of Formula (IVa) or Formula (Va), or a mixture of Formula (IVa) and Formula (Va), the PEG group has an average molecular weight of about 30 kDa.

In any of the embodiments described herein, the structure of Formula (I) has the structure of Formula (IVa) or Formula (Va), or is a mixture of Formula (IVa) and Formula (Va). In some embodiments, the structure of Formula (I) has the structure of Formula (IVa). In some embodiments, the structure of Formula (I) has the structure of Formula (Va). In some embodiments, the structure of Formula (I) is a mixture of Formula (IVa) and Formula (Va).

式 (XII)；

式 (XIII)； 其中： n是整數，使得-(OCH ₂CH ₂) _n-OCH ₃具有約25 kDa-35 kDa的分子量； q是1、2或3；以及 波浪線表示與SEQ ID NO: 1中未被替代的胺基酸殘基的共價鍵。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein amino acid residue P64 is represented by formula (XII) or formula (XIII), or formula (XII) and formula (XIII) ) for the structural substitution of mixtures:

Formula (XII);

Formula (XIII); wherein: n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; and the wavy line represents the same as SEQ ID NO: Covalent bond to the unsubstituted amino acid residue in 1.

In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), q is 1. In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), q is 2. In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), q is 3.

In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has an molecular weight.

In any of the embodiments described herein, the structure of Formula (I) has the structure of Formula (XII) or Formula (XIII), or is a mixture of Formula (XII) and Formula (XIII). In some embodiments, the structure of Formula (I) has the structure of Formula (XII). In some embodiments, the structure of Formula (I) has the structure of Formula (XIII). In some embodiments, the structure of Formula (I) is a mixture of Formula (XII) and Formula (XIII).

式 (XIIa)；

式 (XIIIa)； 其中： n是整數，使得-(OCH ₂CH ₂) _n-OCH ₃具有約25 kDa-35 kDa的分子量；以及 波浪線表示與SEQ ID NO: 1中未被替代的胺基酸殘基的共價鍵。 In some embodiments of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII), q is 1, the structure of formula (XII) is the structure of formula (XIIa), and the formula ( The structure of XIII) is the structure of formula (XIIIa):

Formula (XIIa);

Formula (XIIIa); wherein: n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 25 kDa-35 kDa; and the wavy line represents the unsubstituted amino group in SEQ ID NO: 1 Covalent bonds with acid residues.

In some embodiments of Formula (XIIa) or Formula (XIIIa), or a mixture of Formula (XIIa) and Formula (XIIIa), n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has an molecular weight.

In any of the embodiments described herein, the structure of Formula (I) has the structure of Formula (XIIa) or Formula (XIIIa), or is a mixture of Formula (XIIa) and Formula (XIIIa). In some embodiments, the structure of Formula (I) has the structure of Formula (XIIa). In some embodiments, the structure of Formula (I) has the structure of Formula (XIIIa). In some embodiments, the structure of Formula (I) is a mixture of Formula (XIIa) and Formula (XIIIa).

式 (XIV)；

式 (XV)； 其中： m是0至20的整數； p是0至20的整數； n是整數，使得PEG基團具有約25 kDa-35 kDa的平均分子量；以及 波浪線表示與SEQ ID NO: 1中未被替代的胺基酸殘基的共價鍵。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein amino acid residue P64 is represented by formula (XIV) or formula (XV), or formula (XIV) and formula (XV) ) for the structural substitution of mixtures:

Formula (XIV);

Formula (XV); wherein: m is an integer from 0 to 20; p is an integer from 0 to 20; n is an integer such that the PEG group has an average molecular weight of about 25 kDa-35 kDa; : Covalent bond to the unsubstituted amino acid residue in 1.

In some embodiments of Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV), n is an integer such that the PEG group has an average molecular weight of about 30 kDa.

In some embodiments, m is an integer from 0-15. In some embodiments, m is an integer from 0-10. In some embodiments, m is an integer from 0-5. In some embodiments, m is an integer from 1-5. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5.

In some embodiments, p is an integer from 0-15. In some embodiments, p is an integer from 0-10. In some embodiments, p is an integer from 0-5. In some embodiments, p is an integer from 1-5. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5.

In some embodiments, m and p are each 2.

In any of the embodiments described herein, the structure of Formula (I) has the structure of Formula (XIV) or Formula (XV), or is a mixture of Formula (XIV) and Formula (XV). In some embodiments, the structure of Formula (I) has the structure of Formula (XIV). In some embodiments, the structure of Formula (I) has the structure of Formula (XV). In some embodiments, the structure of Formula (I) is a mixture of Formula (XIV) and Formula (XV).

式 (XVI)；

式 (XVII)； 其中： m是0至20的整數； n是整數，使得PEG基團具有約25 kDa-35 kDa的平均分子量；以及 波浪線表示與SEQ ID NO: 1中未被替代的胺基酸殘基的共價鍵。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein amino acid residue P64 is represented by formula (XVI) or formula (XVII), or formula (XVI) and formula (XVII) ) for the structural substitution of mixtures:

Formula (XVI);

Formula (XVII); wherein: m is an integer from 0 to 20; n is an integer such that the PEG group has an average molecular weight of about 25 kDa-35 kDa; and the wavy line represents the same as the unsubstituted amine in SEQ ID NO: 1 Covalent bonds between amino acid residues.

In some embodiments of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), n is an integer such that the PEG group has an average molecular weight of about 30 kDa.

In some embodiments, m is an integer from 0-15. In some embodiments, m is an integer from 0-10. In some embodiments, m is an integer from 0-5. In some embodiments, m is an integer from 1-5. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5.

In any of the embodiments described herein, the structure of formula (I) has the structure of formula (XVI) or formula (XVII), or is a mixture of formula (XVI) and formula (XVII). In some embodiments, the structure of Formula (I) has the structure of Formula (XVI). In some embodiments, the structure of Formula (I) has the structure of Formula (XVII). In some embodiments, the structure of Formula (I) is a mixture of Formula (XVI) and Formula (XVII). bonding chemistry

In some embodiments, the IL-2 conjugates described herein can be prepared by a conjugation reaction comprising a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises the reaction of an azide with an alkyne ("click" reaction). In some embodiments, the ligation reactions described herein include the reactions outlined in Scheme I, wherein X is the unnatural amino acid at position P64 of SEQ ID NO: 1. Process I.

In some embodiments, the engaging moiety comprises a PEG group as described herein. In some embodiments, reactive groups include alkynes or azides.

In some embodiments, the ligation reactions described herein include the reactions outlined in Scheme II, wherein X is the unnatural amino acid at position P64 of SEQ ID NO: 1. Process II.

In some embodiments, the ligation reactions described herein include the reactions outlined in Scheme III, wherein X is the unnatural amino acid at position P64 of SEQ ID NO: 1. Process III.

In some embodiments, the ligation reactions described herein include the reactions outlined in Scheme IV, wherein X is the unnatural amino acid at position P64 of SEQ ID NO: 1. Process IV.

流程 VI.

In some embodiments, the conjugation reactions described herein include an azide moiety (such as contained in a compound derived from N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) The ring between the azide moiety in the protein of the amino acid residue of ) and a strained cyclic alkyne (such as a strained cyclic alkyne derived from DBCO, which is a chemical moiety containing a dibenzocyclooctyne group) Addition reaction. PEG groups containing DBCO moieties are commercially available or can be prepared by methods known to those of ordinary skill in the art. Exemplary reactions are shown in Schemes V and VI. Process V.

Process VI.

Ligation reactions described herein, such as click reactions, can generate a single positional isomer or a mixture of positional isomers. In some cases, the ratio of positional isomers is about 1:1. In some cases, the ratio of positional isomers is about 2:1. In some cases, the ratio of positional isomers is about 1.5:1. In some cases, the ratio of positional isomers is about 1.2:1. In some cases, the ratio of positional isomers is about 1.1:1. In some cases, the ratio of positional isomers is greater than 1:1. IL-2 polypeptide production

In some instances, the IL-2 conjugates described herein containing natural amino acid mutations or non-natural amino acid mutations are recombinantly produced or chemically synthesized. In some cases, the IL-2 conjugates described herein are produced recombinantly, eg, by host cell systems or in cell-free systems.

In some cases, IL-2 conjugates are produced recombinantly by host cell systems. In some cases, the host cell is a eukaryotic cell (eg, a mammalian cell, an insect cell, a yeast cell, or a plant cell) or a prokaryotic cell (eg, a gram-positive or gram-negative bacterium). In some cases, the eukaryotic host cell is a mammalian host cell. In some cases, the mammalian host cell is a stable cell strain, or a cell strain that has incorporated the genetic material of interest into its own genome and has the ability to express the products of the genetic material after multiple generations of cell division. In other cases, the mammalian host cell is a transient cell strain, or a cell strain that has not incorporated the genetic material of interest into its own genome and has no ability to express the products of the genetic material after multiple generations of cell division.

Exemplary mammalian host cells include 293T cell lines, 293A cell lines, 293FT cell lines, 293F cells, 293H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™- In™-CV-1 cell line, Flp-In™-Jurkat cell line, FreeStyle™ 293-F cell, FreeStyle™ CHO-S cell, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cell, T -REx™ Jurkat cell line, Per.C6 cell line, T-REx™-293 cell line, T-REx™-CHO cell line and T-REx™-HeLa cell line.

In some embodiments, the eukaryotic host cell is an insect host cell. Exemplary insect host cells include Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expressSF+® cells.

In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris ( K. phaffii ) yeast strains, such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strains, Such as INVSc1.

In some embodiments, the eukaryotic host cell is a plant host cell. In some cases, plant cells include cells from algae. Exemplary plant cell lines include lines from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2™ , Stbl3™ or Stbl4™.

In some cases, suitable polynucleic acid molecules or vectors for producing IL-2 polypeptides described herein include any suitable vector derived from a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors from bacterial (eg, E. coli), insect, yeast (eg, Pichia pastoris, S. phaaffia), algal, or mammalian sources. Bacterial vectors include, for example, pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC or pTAC-MAT-2.

Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVPLAG1393 M139, MV1 vectors (such as FLAG1 or pPolh-MAT 2) or a MAT vector (such as pPolh-MAT1 or pPolh-MAT2).

Yeast vectors include, for example, Gateway® pDEST ^™ 14 vector, Gateway® pDEST ^™ 15 vector, Gateway® ^{pDEST ™} 17 vector, Gateway® ^{pDEST ™} 24 vector, Gateway® ^pYES - ^DEST52 vector, pBAD- ^{DEST49 Gateway®} destination vector, pAO815 Pichia Saccharomyces vector, pFLD1 Pichia pastoris (Phiaffia chordii) vector, pGAPZA, B and C Pichia pastoris (Phiaffia chondria) vector, pPIC3.5K Pichia sp. vector, pPIC6 A, B, and C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, and C yeast vector, or pYES3/CT yeast vector.

Algal vectors include, for example, pChlamy-4 vectors or MCS vectors.

Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV7.1, pFLAG-CMV20, p3xFLAG-Myc-CMV24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV3 or pBICEP-CMV4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14. pFLAG-Myc-CMV22, p3xFLAG-Myc-CMV26, pBICEP-CMV1 or pBICEP-CMV2.

In some instances, cell-free systems are used to produce the IL-2 polypeptides described herein. In some cases, cell-free systems comprise a mixture of cytosolic and/or nuclear components from cells and are suitable for in vitro nucleic acid synthesis. In some cases, cell-free systems utilize prokaryotic cellular components. In other cases, cell-free systems utilize eukaryotic cellular components. Nucleic acid synthesis is obtained in cell-free systems based on eg Drosophila cells, Xenopus eggs, Archaea or HeLa cells. Exemplary cell-free systems include E. coli S30 Extraction System, E. coli T7 S30 System or PUREExpress®, XPressCF and XpressCF+.

Cell-free translation systems variously comprise components such as plastids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperones, translation initiation and elongation factors, natural and/or unnatural amino acids and/ or other components for protein expression. Such components are optionally modified to increase yield, increase synthesis rate, increase protein product fidelity, or incorporate unnatural amino acids. In some embodiments, the cytokines described herein are synthesized using the cell-free translation systems described in US 8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245; or US 8,778,631, each of which discloses Incorporated herein by reference. In some embodiments, the cell-free translation system comprises modified release factors, or even removes one or more release factors from the system. In some embodiments, the cell-free translation system comprises reduced protease concentrations. In some embodiments, the cell-free translation system comprises a modified tRNA with reassigned codons encoding unnatural amino acids. In some embodiments, the synthetases described herein are used in a cell-free translation system for incorporation of unnatural amino acids. In some embodiments, the tRNA is preloaded with an unnatural amino acid using enzymatic or chemical means prior to adding the tRNA to the cell-free translation system. In some embodiments, the components used in the cell-free translation system are obtained from modified organisms such as modified bacteria, yeast or other organisms.

In some embodiments, the IL-2 polypeptide is produced in a circularly switched form via an expressed host system or via a cell-free system. Production of cytokine polypeptides comprising unnatural amino acids

Orthogonal or amplified genetic codes may be used in the present disclosure, wherein one or more specific codons present in the nucleic acid sequence of the IL-2 polypeptide are assigned to encode an unnatural amino acid such that it can be It is genetically incorporated into IL-2 by crossing a tRNA synthetase/tRNA pair. Orthogonal tRNA synthetase/tRNA pairs are capable of loading a tRNA with an unnatural amino acid and are capable of incorporating the unnatural amino acid into a polypeptide chain in response to the codon.

In some instances, the codon is a codon amber, ocher, opal, or a quadruple codon. In some cases, the codons correspond to orthogonal tRNAs that will be used to carry the unnatural amino acid. In some instances, the codon is an amber codon. In other cases, the codons are orthogonal codons.

In some cases, the codons are quadruplet codons, which can be decoded by the orthogonal ribosomal ribo-Q1. In some cases, quadruplet codons are as described in: Neumann et al., "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome," Nature , 464 (7287): 441-444 (2010), Its disclosure is incorporated herein by reference.

In some cases, the codons used in the present disclosure are recoded codons, eg, synonymous codons or rare codons replaced by alternative codons. In some cases, codons were recoded as described in Napolitano et al ., "Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli ," PNAS , 113 (38): E5588-5597 (2016), which The disclosure is incorporated herein by reference. In some cases, recoded codons are as described in: Ostrov et al., "Design, synthesis, and testing toward a 57-codon genome," Science 353 (6301): 819-822 (2016), which discloses The contents are incorporated herein by reference.

In some instances, the use of a non-natural nucleic acid results in the incorporation of one or more non-natural amino acids into IL-2. Exemplary unnatural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C ), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2 of adenine and guanine -Propyl derivatives and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine , 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-sulfanyl , 8-hydroxy and other 8-substituted adenine and guanine, 5-halo (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methyl Guanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deaza adenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5 - propynyluracil, 5-propynylcytosine, 5-methylcytosine, those molecules that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated Nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propane Alkynecytosine. 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives of adenine and guanine and others Alkyl derivatives, 2-propyl derivatives of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5 -Halocytosine, 5-propynyl (-C≡C-CH ₃ )uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6- Azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-sulfanyl, 8-Hydroxy and other 8-substituted adenine and guanine, 5-halo (especially 5-bromo), 5-trifluoromethyl, other 5-substituted uracil and cytosine, 7-methylguanine Purine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine Purine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidine, phenoxazine cytidine ([5,4-b][l,4]benzoxazin-2(3H)-one) , phenothiazine cytidine (1H-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamp, phenothiazine cytidine (eg 9- ( 2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), cytazidine (2H-pyrimido[4, 5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), Those in which the purine or pyrimidine bases are replaced by other heterocycles, 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine , bromouracil, 5-chlorocytosine, chlorocytosine, cyclic cytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine , hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil and 5-iodouracil, 2-amino-adenine, 6-thio- Guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, and described in U.S. Patent Nos. 3,687,808; 4,845,205; 4,910,300 ; 4,948,882; 5,093,232; 5,130,302; 5,134,066; 5,175,273; 08；5,502,177；5,525,711；5,552,540；5,587,469；5,594,121；5,596,091；5,614,617；5,645,985；5,681,941；5,750,692；5,763,588；5,830,653和6,005,096；WO 99/62923；Kandimalla等人, (2001) Bioorg. Med. Chem. 9:807- 813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, eds., John Wiley & Sons, 1990, 858- 859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15 , Antisense Research and Applications, Crooke and Lebleu eds., CRC Press, 1993, 273-288. Additional base modifications can be found in, for example, US Pat. No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, pp. 289-302 , Crooke and Lebleu eds., CRC Press, 1993; the disclosure of each of which is incorporated herein by reference.

Non-natural nucleic acids comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and in some cases, the nucleic acids include a One or several heterocyclic bases. For example, in some cases, heterocyclic bases include uracil-5-yl, cytosine-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanine- 8-yl, 4-aminopyrrolo[2.3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2, 3-d]pyrimidin-5-yl, 2-amino-4 -Oxopyrrolo[2.3-d]pyrimidin-3-yl, wherein the purine is attached to the nucleic acid via the 9-position, the pyrimidine via the 1-position, the pyrrolopyrimidine via the 7-position and the pyrazolopyrimidine via the 1-position sugar part.

In some embodiments, the nucleotide analogs are also modified in the phosphate moiety. Modified phosphate moieties include, but are not limited to, those that are modified at the junction between two nucleotides, and contain, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphorotriphosphate esters, aminoalkylphosphonates, methyl and other alkylphosphonates (including 3'-alkylenephosphonates) and chiral phosphonates, phosphinates, phosphoramidates (including 3'- '-Aminophosphonoamido and aminoalkylphosphoramido, thiocarboamidophosphate), thionoalkyl phosphonate, thionoalkyl phosphate triester, and borane phosphate. It is understood that these phosphate or modified phosphate linkages between two nucleotides are via 3'-5' linkages or 2'-5' linkages, and that the linkages contain reverse polarity, such as 3' -5' to 5'-3' or 2'-5' to 5'-2'. Also included are the various salts, mixed salts and free acid forms. A number of U.S. patents teach how to make and use nucleotides containing modified phosphates, and include, but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,399,676；5,405,939；5,453,496；5,455,233；5,466,677；5,476,925；5,519,126；5,536,821；5,541,306；5,550,111；5,563,253；5,571,799；5,587,361；和5,625,050；其每一個的揭示內容藉由引用併入本文。

In some embodiments, non-natural nucleic acids include 2',3'-dideoxy-2',3'-didehydro-nucleosides (PCT/US2002/006460), 5'-substituted DNA and RNA derivatives (PCT/US2011/033961; Saha et al., J. Org Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020; PCT PCT/JP2003/002342; PCT/JP2004/013216; PCT/JP2005/020435; PCT/JP2006/315479; PCT/JP2006/324484; PCT/JP2009/056718; PCT/JP2006/ 5'-substituted monomers of monophosphates with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337); the disclosure of each is borrowed from Incorporated herein by reference.

In some embodiments, the non-natural nucleic acid includes modifications at the 5'-position and the 2'-position of the sugar ring (PCT/US94/02993), such as 5'- _CH2 -substituted 2'-O-protected Nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem. 1999, 10, 921-924). In some cases, non-natural nucleic acids include amide-linked nucleoside dimers that have been prepared for incorporation into oligonucleotides, wherein the 3'-linked nucleosides in the dimer (5' to 3' ) contains 2'-OCH ₃ and 5'-(S)-CH ₃ (Mesmaeker et al., Synlett, 1997, 1287-1290). Non-natural nucleic acids may include 2'-substituted 5'- _CH2 (or O) modified nucleosides (PCT/US92/01020). Unnatural nucleic acids can include 5'-methylene phosphonate DNA and RNA monomers, and dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703-705; and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Non-natural nucleic acids may include 5'-phosphonate monomers with 2'-substituents (US 2006/0074035) and other modified 5'-phosphonate monomers (WO 1997/35869). Non-natural nucleic acids may include 5'-modified methylene phosphonate monomers (EP614907 and EP629633). Non-natural nucleic acids may include analogues of 5' or 6'-phosphonate ribonucleosides containing hydroxyl groups at the 5' and/or 6' positions (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786 ; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem. , 1976, 19(8), 1029-1033). Non-natural nucleic acids may include 5'-phosphonate deoxyribonucleoside monomers and dimers with 5'-phosphate groups (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Non-natural nucleic acids may include nucleosides with 6'-phosphonate groups (where the 5' and/or 6' positions are unsubstituted or replaced with thio-tert-butyl (SC(CH ₃ ) ₃ ) (and analogs); methyleneamine (CH ₂ NH ₂ ) (and its analogs) or cyano (CN) (and its analogs) substitutions (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al., J. Med. Chem., 1976, 19, 1371-1377; Geze et al., J. Am. Chem. Soc, 1983, 105(26), 7638-7640; and Hampton et al., J. Am. Chem. Soc, 1973, 95(13), 4404-4414). The disclosure of each reference listed in this paragraph is incorporated herein by reference.

In some embodiments, the non-natural nucleic acid also includes modifications of the sugar moiety. In some cases, nucleic acids contain one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, the nucleic acid comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, addition of substituents (including 5' and/or 2'substituents; bridging of two ring atoms to form a bicyclic nucleic acid (BNA); (R ₁ )(R ₂ ) replaces the ribosyl epoxy atom (R = H, C ₁ -C ₁₂ alkyl or protecting group); and combinations thereof. Examples of chemically modified sugars can be found in WO 2008/101157 , US 2005/0130923 and WO 2007/134181, the disclosures of each of which are incorporated herein by reference.

In some cases, a modified nucleic acid comprises a modified sugar or sugar analog. Thus, in addition to ribose and deoxyribose, the sugar moiety may be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose or sugar "analogue" Cyclopentyl. The sugar may be in the pyranosyl or furanosyl form. The sugar moiety may be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar may be attached to the corresponding hetero in the [α] or [β] anomeric configuration. Cyclic bases. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amine-RNA/DNA chimeras . For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-glycol-like ribonucleosides. The preparation of these sugars or sugar analogs and the corresponding "nucleosides" in which such sugars or analogs are attached to heterocyclic bases (nucleic acid bases) is known. Sugar modifications can also be made and combined with other modifications.

Modifications of sugar moieties include natural modifications of ribose and deoxyribose sugars as well as non-natural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkyne or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl groups. 2' sugar modification also includes but not limited to -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , —O(CH ₂ ) _n ONH ₂ , and —O(CH ₂ ) _n ON[(CH ₂ )n CH ₃ )] ₂ , where n and m are 1 to about 10.

Other modifications at the 2' position include, but are not limited to: _C1 to _C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH _3. OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkane Amino groups, polyalkylamine groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides or for improving oligonucleotides A group with the pharmacodynamic properties of an acid, and other substituents with similar properties. Also at other positions of the sugar (especially at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in oligonucleotides at the 3' terminal or 2'-5' linked oligonucleotides) Make similar modifications. Modified sugars also include those sugars that contain modifications such as _CH2 and S at the bridge epoxy. Nucleotide sugar analogs may also have sugar mimics, such as a cyclobutyl moiety in place of a pentofuranosyl sugar. A number of U.S. patents teach the preparation of such modified sugar structures and detail and describe a series of base modifications, such as U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; ；5,514,785；5,519,134；5,567,811；5,576,427；5,591,722；5,597,909；5,610,300；5,627,053；5,639,873；5,646,265；5,658,873；5,670,633；4,845,205；5,130,302；5,134,066；5,175,273；5,367,066；5,432,272；5,457,187；5,459,255；5,484,908；5,502,177；5,525,711；5,552,540；5,587,469 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,700,920, the disclosures of each of which are incorporated herein by reference.

Examples of nucleic acids with modified sugar moieties include, but are not limited to, those containing 5''-vinyl, 5''-methyl (R or S), 4'-S, 2'-F, 2' _- OCH and Nucleic acid with 2'-O(CH ₂ ) ₂ OCH ₃ substituent. The substituent at the 2' position may also be selected from allyl, amine, azido, thio, O-allyl, O-(C ₁ -C ₁₀ alkyl), OCF ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ -ON(R _m )(R _n ), and O-CH ₂ -C(=O)-N(R _m )(R _n ), wherein R _m and R _n are each are independently H or substituted or unsubstituted C ₁ -C ₁₀ alkyl.

In certain embodiments, the nucleic acids described herein include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, wherein the bridge comprises a 4' to 2' bicyclic nucleic acid. Examples of such 4' to 2' bicyclic nucleic acids include, but are not limited to, one of the following formulas: 4'-(CH ₂ )-O-2'(LNA);4'-(CH ₂ )-S-2'; 4 '-(CH ₂ ) ₂ -O-2'(ENA);4'-CH(CH ₃ )-O-2' and 4'-CH(CH ₂ OCH ₃ )-O-2' and their analogs ( See US Patent No. 7,399,845); 4'-C(CH ₃ )(CH ₃ )-O-2' and its analogs (see WO 2009/006478, WO 2008/150729, US 2004/0171570, US Patent No. 7,427,672, Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134, and WO 2008/154401). See also eg: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000 , 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al. , J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol, 2001, 8 , 1-7；Oram等人, Curr. Opinion Mol. Ther., 2001, 3, 239-243；美國專利號4,849,513；5,015,733；5,118,800；5,118,802；7,053,207；6,268,490；6,770,748；6,794,499；7,034,133；6,525,191；6,670,461； and 7,399,845; International Publication Nos. WO 2004/106356, WO 1994/14226, WO 2005/021570, WO 2007/090071, and WO 2007/134181; U.S. Patent Publication Nos. US 2004/0171570, US 2007/0287831, and US 20908/080; U.S. Provisional Application Nos. 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and International Application Nos. PCT/US2008/064591, PCT US2008/066154, PCT US2008/ 068922 and PCT/DK98/00393. The disclosure of each reference listed in this paragraph is incorporated herein by reference.

In certain embodiments, nucleic acids comprise linked nucleic acids. Nucleic acids can be linked together using any internucleic acid linkage. Two main classes of internucleic acid linkers are defined by the presence or absence of phosphorus atoms. Representative phosphorus-containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative phosphorus-free internucleic acid linking groups include, but are not limited to, methylenemethylimino ( _-CH2 -N( _CH3 ) _-O -CH2-), thiodiester (-OC( O)-S-), thiourethanes (-OC(O)(NH)-S-); siloxanes (-O-Si(H) ₂ -O-); and N,N* - Dimethylhydrazine ( _-CH2 -N( _CH3 )-N( _CH3 )). In certain embodiments, internucleic acid linkages with chiral atoms can be prepared as racemic mixtures as separate enantiomers, eg, alkylphosphonates and phosphorothioates. A non-natural nucleic acid can contain individual modifications. A non-natural nucleic acid may contain multiple modifications within one of the portions or between different portions.

Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate (bridged or unbridged), phosphotriester, phosphorodithioate, phosphorodithioate (phosphodithioate) and borane phosphate, and may be used in any combination. Other non-phosphate linkages can also be used.

In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate, and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity on the modified nucleic acid and/or Enhance its in vivo stability.

In some cases, phosphorus derivatives (or modified phosphate groups) are attached to sugar or sugar analog moieties and can be monophosphate, diphosphate, triphosphate, alkylphosphonate, thio Phosphate, phosphorodithioate, phosphoroamidate, etc. Exemplary polynucleotides containing modified phosphate linkages or non-phosphate linkages can be found in: Peyrottes et al., 1996, Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24: 2318-2323; Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, “Oligonucleotide Analogs: an Overview” in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, eds.) John Wiley and Sons, New York, NY; Zon, 1993, “Oligonucleoside Phosphorothioates” in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; U.S. Patent No. 5,453,496; and Micklefield, 2001, Curr. Med. Chem. 8: 1157-1179; disclosures of each The contents are incorporated herein by reference.

In some cases, backbone modifications include replacing phosphodiester linkages with alternative moieties such as anionic groups, neutral groups, or cationic groups. Examples of such modifications include: anionic internucleoside linkages; N3' to P5' phosphoramidate modifications; borane phosphate DNA; pro-oligonucleotides; neutral internucleoside linkages such as methylphosphonate; Amide-linked DNA; methylene (methylimino) linkages; formacetal and thioformal linkages; sulfonyl-containing backbones; N-morpholin-based oligomers; peptides nucleic acid (PNA); and positively charged deoxyribonucleic acid guanidine (DNG) oligomers (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179, the disclosure of which is incorporated herein by reference). A modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications (eg, a combination of phosphate linkages, such as a combination of phosphodiester and phosphorothioate linkages).

Substituents for phosphate esters include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatoms or heterocycles Internucleoside linkage. These include those with: N-morpholinyl linkages (formed in part from the sugar moieties of nucleosides); siloxane backbones; sulfide, sulfide, and sulfide backbones; formylacetyl and thioformyl Acetyl skeletons; methyleneformylacetyl and thioformylacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazine skeletons; ester and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and _CH moieties. A number of U.S. patents disclose how to make and use these types of phosphate ester substitutes and include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; ；5,470,967；5,489,677；5,541,307；5,561,225；5,596,086；5,602,240；5,610,289；5,602,240；5,608,046；5,610,289；5,618,704；5,623,070；5,663,312；5,633,360；5,677,437；和5,677,439。 It is also understood that in nucleotide substitutions both the sugar and phosphate moieties of the nucleotide may be replaced, for example by an amide-type linkage (aminoethylglycine) (PNA). US Patent Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to attach other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance eg cellular uptake. A conjugate may be chemically linked to the nucleotide or nucleotide analog. Such conjugates include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), bile acids (Manoharan et al., Bioorg. Med. Chem. . Let., 1994, 4, 1053-1060), thioethers, for example, hexyl-S-trityl mercaptan (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as , dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or l-di-O-hexadecyl-rac-glycerol-SH-triethylammonium phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides , 1995, 14, 969-973), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).許多美國專利傳授了此類接合物的製備，並且所述美國專利包括但不限於美國專利號4,828,979；4,948,882；5,218,105；5,525,465；5,541,313；5,545,730；5,552,538；5,578,717, 5,580,731；5,580,731；5,591,584；5,109,124；5,118,802； 5,138,045；5,414,077；5,486,603；5,512,439；5,578,718；5,608,046；4,587,044；4,605,735；4,667,025；4,762,779；4,789,737；4,824,941；4,835,263；4,876,335；4,904,582；4,958,013；5,082,830；5,112,963；5,214,136；5,082,830；5,112,963；5,214,136；5,245,022；5,254,469；5,258,506； 5,262,536；5,272,250；5,292,873；5,317,098；5,371,241, 5,391,723；5,416,203, 5,451,463；5,510,475；5,512,667；5,514,785；5,565,552；5,567,810；5,574,142；5,585,481；5,587,371；5,595,726；5,597,696；5,599,923；5,599,928和5,688,941。 The disclosure of each reference listed in this paragraph is incorporated herein by reference.

。 示例性非天然鹼基對包括：(d)TPT3-(d)NaM；(d)5SICS-(d)NaM；(d)CNMO-(d)TAT1；(d)NaM-(d)TAT1；(d)CNMO-(d)TPT3；和(d)5FM-(d)TAT1。 In some cases, the unnatural nucleic acid further forms unnatural base pairs. Exemplary unnatural nucleotides capable of forming unnatural DNA or RNA base pairs (UBPs) under in vivo conditions include, but are not limited to, TAT1, dTAT1, 5FM, d5FM, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO , dCNMO and combinations thereof. In some embodiments, non-natural nucleotides include:

. Exemplary unnatural base pairs include: (d)TPT3-(d)NaM; (d)5SICS-(d)NaM; (d)CNMO-(d)TAT1; (d)NaM-(d)TAT1; ( d) CNMO-(d) TPT3; and (d) 5FM-(d) TAT1.

。 Additional examples of non-natural nucleotides capable of forming non-natural UBPs that can be used to make the IL-2 conjugates disclosed herein can be found in Dien et al., J Am Chem Soc., 2018, 140:16115-16123; Feldman et al. , J Am Chem Soc, 2017, 139:11427-11433; Ledbetter et al., J Am Chem Soc., 2018, 140:758-765; Dhami et al., Nucleic Acids Res. 2014, 42:10235-10244; Malyshev et al. People, Nature, 2014, 509:385-388; Betz et al., J Am Chem Soc., 2013, 135:18637-18643; Lavergne et al., J Am Chem Soc. 2013, 135:5408-5419; and Malyshev et al. Proc Natl Acad Sci USA, 2012, 109:12005-12010; the disclosure of each of which is incorporated herein by reference. In some embodiments, non-natural nucleotides include:

.

其中R ₂選自氫、烷基、烯基、炔基、甲氧基、甲硫醇、甲烷硒基、鹵素、氰基和疊氮基；並且 波浪線指示與核糖基或2’-去氧核糖基的鍵，其中核糖基或2’-去氧核糖基部分的5’-羥基為游離形式，連接至單磷酸酯、二磷酸酯、三磷酸酯、α-硫代三磷酸酯、β-硫代三磷酸酯或γ-硫代三磷酸酯基團，或包含在RNA或DNA中或者RNA類似物或DNA類似物中。 In some embodiments, non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein can be derived from compounds of the formula:

Wherein R is selected from _hydrogen , alkyl, alkenyl, alkynyl, methoxy, methylmercaptan, methaneselenoyl, halogen, cyano and azido; Ribosyl bond, where the 5'-hydroxyl of the ribose or 2'-deoxyribose moiety is in free form, attached to a monophosphate, diphosphate, triphosphate, alpha-thiotriphosphate, beta- Thiotriphosphate or gamma-thiotriphosphate groups, either contained in RNA or DNA or RNA analogs or DNA analogs.

其中： 每個X獨立地是碳或氮； 當X是氮時R ₂不存在，而當X是碳時，R ₂是存在的且獨立地是氫、烷基、烯基、炔基、甲氧基、甲硫醇、甲烷硒基、鹵素、氰基或疊氮基； Y是硫、氧、硒或二級胺； E是氧、硫或硒；並且 波浪線指示與核糖基、去氧核糖基或二去氧核糖基部分或其類似物的鍵合點，其中所述核糖基、去氧核糖基或二去氧核糖基部分或其類似物是游離形式，連接至單磷酸酯、二磷酸酯、三磷酸酯、α-硫代三磷酸酯、β-硫代三磷酸酯或γ-硫代三磷酸酯基團，或包含在RNA或DNA中或者RNA類似物或DNA類似物中。 In some embodiments, non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein can be derived from compounds of the formula:

wherein: each X is independently carbon or nitrogen _; R is absent when X is nitrogen, and R is present when X is carbon and is independently _hydrogen , alkyl, alkenyl, alkynyl, methyl Oxygen, methylthiol, methaneselenyl, halogen, cyano, or azido; Y is sulfur, oxygen, selenium, or a secondary amine; E is oxygen, sulfur, or selenium; and the wavy line indicates the same as ribose, deoxy Ribosyl or dideoxyribosyl moiety or analog thereof, wherein said ribose, deoxyribose or dideoxyribose moiety or analog thereof is in free form, attached to a monophosphate, di Phosphate, triphosphate, alpha-thiotriphosphate, beta-thiotriphosphate or gamma-thiotriphosphate groups, either contained in RNA or DNA or RNA analogs or DNA analogs.

In some embodiments, each X is carbon. In some embodiments, at least one X is carbon. In some embodiments, one X is carbon. In some embodiments, at least two X's are carbon. In some embodiments, both X's are carbon. In some embodiments, at least one X is nitrogen. In some embodiments, one X is nitrogen. In some embodiments, at least two X's are nitrogen. In some embodiments, both X's are nitrogen.

In some embodiments, Y is sulfur. In some embodiments, Y is oxygen. In some embodiments, Y is selenium. In some embodiments, Y is a secondary amine.

In some embodiments, E is sulfur. In some embodiments, E is oxygen. In some embodiments, E is selenium.

_In some embodiments, R2 is present when X is carbon. ^In some embodiments, R2 is absent when X is nitrogen. In some embodiments, each R ₂ , where present, is hydrogen. In some embodiments, R ₂ is alkyl, such as methyl, ethyl or propyl. In some embodiments, R ₂ is alkenyl, such as —CH ₂ ═CH ₂ . In some embodiments, R ₂ is alkynyl, such as ethynyl. In some embodiments, R ₂ is methoxy. In some embodiments, R ₂ is methyl mercaptan. In some embodiments, R ₂ is methaneselenoyl. _In some embodiments, R2 is halogen, such as chlorine, bromine or fluorine. In some embodiments, R ₂ is cyano. In some embodiments, R ₂ is azido.

In some embodiments, E is sulfur, Y is sulfur, and each X is independently carbon or nitrogen. In some embodiments, E is sulfur, Y is sulfur, and each X is carbon.

、

、

、

、

、

、

、

、

、

、

和

。在一些實施例中，可以用於製備本文公開的IL-2接合物的非天然核苷酸包括

、

、

、

、

、

、

、

、

、

、

和

或其鹽。 In some embodiments, non-natural nucleotides that can be used to make the IL-2 conjugates disclosed herein can be derived from

,

,

,

,

,

,

,

,

,

,

with

. In some embodiments, non-natural nucleotides that can be used to make the IL-2 conjugates disclosed herein include

,

,

,

,

,

,

,

,

,

,

with

or its salt.

In some embodiments, unnatural base pairs result in unnatural amino acids, as described in: Dumas et al ., "Designing logical codon reassignment - Expanding the chemistry in biology," Chemical Science , 6 : 50-69 ( 2015), the disclosures of which are incorporated herein by reference.

In some embodiments, non-natural amino acids are incorporated into cytokines (eg, IL polypeptides) by synthetic codons comprising non-natural nucleic acids. In some cases, unnatural amino acids were incorporated into cytokines by orthogonal modified synthetase/tRNA pairs. Such orthogonal pairs comprise natural synthetases capable of loading a non-natural tRNA with a non-natural amino acid while minimizing a) other endogenous amino acids on the non-natural tRNA and b) non-natural amine groups Acid loading on other endogenous tRNAs. Such orthogonal pairs comprise tRNAs capable of loading by non-natural synthetases while avoiding loading of a) other endogenous amino acids by endogenous synthetases. In some embodiments, such pairs are identified from various organisms such as bacterial, yeast, archaeal or human sources. In some embodiments, an orthogonal synthetase/tRNA pair comprises components from a single organism. In some embodiments, an orthogonal synthetase/tRNA pair comprises components from two different organisms. In some embodiments, the orthogonal synthetase/tRNA pair comprises components that facilitate translation of two different amino acids prior to modification. In some embodiments, the orthogonal synthetase is a modified alanine synthase. In some embodiments, the orthogonal synthetase is a modified arginine synthase. In some embodiments, the orthogonal synthetase is a modified asparagine synthetase. In some embodiments, the orthogonal synthetase is a modified aspartate synthetase. In some embodiments, the orthogonal synthetase is a modified cysteine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamate synthase. In some embodiments, the orthogonal synthetase is a modified alanine glycine. In some embodiments, the orthogonal synthetase is a modified histidine synthase. In some embodiments, the orthogonal synthetase is a modified leucine synthase. In some embodiments, the orthogonal synthetase is a modified isoleucine synthase. In some embodiments, the orthogonal synthetase is a modified lysine synthase. In some embodiments, the orthogonal synthetase is a modified methionine synthetase. In some embodiments, the orthogonal synthetase is a modified phenylalanine synthase. In some embodiments, the orthogonal synthetase is a modified proline synthase. In some embodiments, the orthogonal synthetase is a modified serine synthetase. In some embodiments, the orthogonal synthetase is a modified threonine synthase. In some embodiments, the orthogonal synthetase is a modified tryptophan synthase. In some embodiments, the orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, the orthogonal synthetase is a modified streptine synthase. In some embodiments, the orthogonal synthetase is a modified phosphoserine synthetase. In some embodiments, the orthogonal tRNA is a modified alanine tRNA. In some embodiments, the orthogonal tRNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartic acid tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamate tRNA. In some embodiments, the orthogonal tRNA is a modified alanine glycine. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA. In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

In some embodiments, an unnatural amino acid is incorporated into a cytokine (eg, IL polypeptide) by an amide (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, but are not limited to, the Methanococcus jannaschii ( Mj-Tyr ) aaRS/tRNA pair, the Escherichia coli TyrRS ( Ec-Tyr )/ B. stearothermophilus tRNA _CUA pair , Escherichia coli LeuRS ( Ec-Leu )/Bacillus stearothermophilus tRNA _CUA pair and pyrrole lysyl-tRNA pair. In some instances, unnatural amino acids are incorporated into cytokines (eg, IL polypeptides) by Mj-Tyr RS/tRNA pairs. Exemplary UAAs that can be incorporated by the Mj-Tyr RS/tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methoxyphenylalanine; meta-substituted tyramine; Amino acid derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boron phenylalanine; and o-nitrobenzyltyrosine.

In some instances, unnatural amino acids are incorporated into cytokines (eg, IL polypeptides) by Ec-Tyr /tRNA _CUA or Ec-Leu /tRNA _CUA pairs. Exemplary UAAs that can be incorporated by Ec-Tyr /tRNA _CUA or Ec-Leu /tRNA _CUA pairs include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O— propargyltyrosine; alpha-aminooctanoic acid, O-methyltyrosine, O-nitrobenzylcysteine; and 3-(naphthalene-2-ylamino)-2-amino- propionic acid.

In some instances, unnatural amino acids are incorporated into cytokines (eg, IL polypeptides) by pyrrolyl-tRNA pairs. In some cases, the PylRS is obtained from an archaea, eg, from a methanogenic archaea. In some instances, the PylRS is obtained from Methanosarcina barkeri , Methanosarcina mazei , or Methanosarcina acetivorans . Exemplary UAAs that can be incorporated via pyrrolyl-tRNA pairs include, but are not limited to, amide and carbamate substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2 -formamido)hexanoic acid, N -ε- _D -prolinyl- _L -lysine and N -ε-cyclopentyloxycarbonyl- _L -lysine; N -ε-acrylyl _-L -lysine; N -ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl] _-L- lysine; and N -ε-(1-methylcycloprop-2-encarbamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein can be prepared by using M. mazei tRNA by M. barkeri pyrrole Lysyl-tRNA synthetase ( Mb PylRS) selectively loads unnatural amino acids such as N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647, the disclosure of which is incorporated herein by reference.

In some instances, unnatural amino acids are incorporated into the cytokines (e.g., IL polypeptides) described herein by synthetases disclosed in US 9,988,619 and US 9,938,516, the disclosures of each of which are incorporated by reference This article.

The host cells into which the constructs or vectors disclosed herein are introduced are cultured or maintained in a suitable medium such that tRNA, tRNA synthetase, and protein of interest are produced. The medium also includes one or more unnatural amino acids such that the protein of interest incorporates the one or more unnatural amino acids. In some embodiments, a nucleoside triphosphate transporter (NTT) from bacteria, plants or algae is also present in the host cell. In some embodiments, the IL-2 conjugates disclosed herein are produced by using NTT-expressing host cells. In some embodiments, the nucleoside triphosphate transporter used in the host cell can be selected from TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana) ), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P. tricornutum), GsNTT (Galdieria sulphuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana) , CtNTT1, CtNTT2 (Chlamydia trachomatis), PamNTT1, PamNTT2 (Protochlamydia amoebophila), CcNTT (Caedibacter caryophilus), RpNTT1 (Rickettsia prowazekii)). In some embodiments, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, the NTT is PtNTT1. In some embodiments, the NTT is PtNTT2. In some embodiments, the NTT is PtNTT3. In some embodiments, the NTT is PtNTT4. In some embodiments, the NTT is PtNTT5. In some embodiments, the NTT is PtNTT6. Other NTTs that can be used are disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; Malyshev et al. Nature 2014 (509(7500), 385-388; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322.

Orthogonal tRNA synthetase/tRNA pairs load the tRNA with an unnatural amino acid and incorporate the unnatural amino acid into a polypeptide chain in response to the codon. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii ( Mj-Tyr ) aaRS/tRNA pair, Escherichia coli TyrRS ( Ec-Tyr )/ B. stearothermophilus tRNA _CUA pair , Escherichia coli LeuRS ( Ec-Leu )/Bacillus stearothermophilus tRNA _CUA pair and pyrrole lysyl-tRNA pair. Other aaRS-tRNA pairs that can be used in accordance with this disclosure include those derived from Methanosarcina mazei, those described in: Feldman et al., J Am Chem Soc., 2018 140:1447-1454; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322; the disclosure of each of which is incorporated herein by reference.

In some embodiments are provided methods of making the IL-2 conjugates disclosed herein in a cell system expressing NTT and tRNA synthetase. In some embodiments described herein, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii, Escherichia coli TyrRS ( Ec-Tyr )/Bacillus stearothermophilus and Methanosarcina mazei. In some embodiments, the NTT is PtNTT1 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( Ec-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT2 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( Ec-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( Ec-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( Ec-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT4 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( Ec-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT5 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( Ec-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT6 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( Ec-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei.

、

、

、

、

和

。在一些實施例中，在雙鏈寡核苷酸中的包含非天然鹼基對的第一和第二非天然核苷酸可以衍生自

和

。在一些實施例中，第一和第二非天然核苷酸的三磷酸酯包括

、

和

、或其鹽。在一些實施例中，第一和第二非天然核苷酸的三磷酸酯包括

、和

、或其鹽。在一些實施例中，源自包含第一非天然核苷酸和第二非天然核苷酸的雙鏈寡核苷酸的mRNA可以包含含有衍生自

、

和

的非天然核苷酸的密碼子。在一些實施例中，馬氏甲烷八疊球菌tRNA可以包含含有非天然核苷酸的反密碼子，所述非天然核苷酸識別包含mRNA的非天然核苷酸的密碼子。馬氏甲烷八疊球菌tRNA中的反密碼子可以包含衍生自

、

、

、

、

和

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸。在一些實施例中，tRNA包含衍生自

的非天然核苷酸。在一些實施例中，tRNA包含衍生自

的非天然核苷酸。在一些實施例中，tRNA包含衍生自

的非天然核苷酸。在一些實施例中，tRNA包含衍生自

的非天然核苷酸。在一些實施例中，tRNA包含衍生自

的非天然核苷酸。在一些實施例中，tRNA包含衍生自

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸，並且tRNA包含衍生自

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸，並且tRNA包含衍生自

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸，並且tRNA包含衍生自

的非天然核苷酸。在一些實施例中，mRNA包含衍生自

的非天然核苷酸，並且tRNA包含衍生自

的非天然核苷酸。將宿主細胞在含有適當營養素的培養基中培養，並補充以下物質：(a) 包含一種或多種非天然鹼基的去氧核糖核苷的三磷酸酯，所述非天然鹼基是編碼具有密碼子的細胞激素基因的一種或多種質體的複製所需的，(b) 包含一種或多種非天然鹼基的核糖核苷的三磷酸酯，所述非天然鹼基是以下的轉錄所需的：(i) 對應於細胞激素的編碼序列並且含有包含一種或多種非天然鹼基的密碼子的mRNA，和 (ii) 含有包含一種或多種非天然鹼基的反密碼子的tRNA，以及 (c) 要摻入目的細胞激素的多肽序列中的一種或多種非天然胺基酸。然後將宿主細胞維持在允許目的蛋白質表現的條件下。 In some embodiments, IL-2 conjugates disclosed herein can be produced in cells (such as E. coli) comprising (a) the nucleotide triphosphate transporter Pt NTT2 (including truncated variants, wherein the full-length protein deletion of the first 65 amino acid residues), (b) plastids containing double-stranded oligonucleotides encoding IL-2 variants with the desired amino acid sequence and containing An unnatural base pair comprising a first unnatural nucleotide and a second unnatural nucleotide to provide a codon at the desired position where an unnatural amino acid will be incorporated, such as N6- ( (2-azidoethoxy)-carbonyl)-L-lysine (AzK), (c) a plastid encoding a tRNA derived from M. mazei and comprising unnatural nucleotides to Putative anticodons (codons for IL-2 variants) are provided in place of their native sequences, and (d) the plasmid encoding the pyrrolysine-tRNA synthetase ( Mb PylRS) derived from Mb PylRS plastid, which may be the same plastid encoding said tRNA or a different plastid. In some embodiments, the cells are further supplemented with deoxyribose triphosphate comprising one or more unnatural bases. In some embodiments, the cells are further supplemented with ribose triphosphate comprising one or more unnatural bases. In some embodiments, the cells are further supplemented with one or more unnatural amino acids, such as N6 -((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the double-stranded oligonucleotide encoding the amino acid sequence of the desired IL-2 variant contains the codon AXC at position 64 of the sequence encoding the protein having SEQ ID NO: 1, where X is an unnatural nucleotide. In some embodiments, the cell further comprises a plastid, which may be a protein expressing plastid or another plastid encoding an orthogonal tRNA gene from M. mazei comprising The AXC-matched anticodon GYT replaces its native sequence, where Y is an unnatural nucleotide that is complementary and may be the same as or different from the unnatural nucleotide in said codon. In some embodiments, the unnatural nucleotide in the codon is different from and complementary to the unnatural nucleotide in the anticodon. In some embodiments, the unnatural nucleotide in the codon is the same as the unnatural nucleotide in the anticodon. In some embodiments, the first and second unnatural nucleotides comprising an unnatural base pair in a double-stranded oligonucleotide can be derived from

,

,

,

,

with

. In some embodiments, the first and second unnatural nucleotides comprising an unnatural base pair in a double-stranded oligonucleotide can be derived from

with

. In some embodiments, the triphosphates of the first and second unnatural nucleotides include

,

with

, or a salt thereof. In some embodiments, the triphosphates of the first and second unnatural nucleotides include

,with

, or a salt thereof. In some embodiments, mRNA derived from a double-stranded oligonucleotide comprising a first unnatural nucleotide and a second unnatural nucleotide may comprise a

,

with

codons for unnatural nucleotides. In some embodiments, the M. mazei tRNA can comprise an anticodon comprising a non-natural nucleotide that recognizes a codon comprising a non-natural nucleotide of the mRNA. Anticodons in the tRNA of M. mazei can contain anticodons derived from

,

,

,

,

with

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

unnatural nucleotides. In some embodiments, the tRNA comprises derived from

unnatural nucleotides. In some embodiments, the tRNA comprises derived from

unnatural nucleotides. In some embodiments, the tRNA comprises derived from

unnatural nucleotides. In some embodiments, the tRNA comprises derived from

unnatural nucleotides. In some embodiments, the tRNA comprises derived from

unnatural nucleotides. In some embodiments, the tRNA comprises derived from

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides, and the tRNA contains derived from

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides, and the tRNA contains derived from

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides, and the tRNA contains derived from

unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides, and the tRNA contains derived from

unnatural nucleotides. The host cells are cultured in medium containing appropriate nutrients and supplemented with: (a) deoxyribonucleoside triphosphates comprising one or more unnatural bases encoding (b) triphosphates of ribonucleosides comprising one or more unnatural bases required for the transcription of: (i) an mRNA corresponding to a coding sequence for a cytokine and containing a codon comprising one or more unnatural bases, and (ii) a tRNA comprising an anticodon comprising one or more unnatural bases, and (c) One or more unnatural amino acids to be incorporated into the polypeptide sequence of the cytokine of interest. The host cells are then maintained under conditions that permit expression of the protein of interest.

The resulting AzK-containing protein expressed can be purified by methods known to those of ordinary skill in the art and then can be allowed to combine with an alkyne such as DBCO comprising a PEG chain having the desired average molecular weight as disclosed herein. reacted under conditions known to the skilled artisan to provide the IL-2 conjugates disclosed herein. Other methods are known to those of ordinary skill in the art, such as those disclosed in: Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; 2019014262; WO 2019014267; WO 2019028419; and WO 2019/028425, the disclosures of each of which are incorporated herein by reference.

The resulting protein expressed comprising one or more unnatural amino acids (e.g., Azk) can be purified by methods known to those of ordinary skill in the art, and then can be allowed to react with alkynes (such as those comprising the amino acids as disclosed herein) having the DBCO) of PEG chains of desired average molecular weight) are reacted under conditions known to those of ordinary skill in the art to provide the IL-2 conjugates disclosed herein. Other methods are known to those of ordinary skill in the art, such as those disclosed in: Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; 2019014262; WO 2019014267; WO 2019028419; and WO 2019/028425, the disclosures of each of which are incorporated herein by reference.

Alternatively, prepared by introducing into a host cell a nucleic acid construct as described herein comprising a tRNA and an amide tRNA synthetase comprising a nucleic acid sequence of interest with one or more in-frame orthogonal (stop) codons IL-2 polypeptides comprising one or more unnatural amino acids. The host cell is cultured in a medium containing appropriate nutrients supplemented with (a) deoxyribonucleoside triphosphates comprising one or more unnatural bases that contribute to a (b) ribonucleoside triphosphates for the mRNA corresponding to Required for transcription: (i) a cytokine sequence containing codons, and (ii) an orthogonal tRNA containing anticodons; and (c) one or more unnatural amino acids. The host cells are then maintained under conditions that permit expression of the protein of interest. The one or more unnatural amino acids are incorporated into the polypeptide chain in response to the unnatural codon. For example, one or more unnatural amino acids are incorporated into the IL-2 polypeptide. Alternatively, two or more unnatural amino acids can be incorporated into the IL-2 polypeptide at two or more sites on the protein.

Once produced in a host cell, the IL-2 polypeptide incorporating the unnatural amino acid can be extracted therefrom by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. IL-2 polypeptides can be purified by standard techniques known in the art, such as preparative ion exchange chromatography, hydrophobic chromatography, affinity chromatography, or any other suitable technique known to those of ordinary skill in the art.

Suitable host cells may include bacterial cells (e.g. E. coli, BL21(DE3)), but most suitable host cells are eukaryotic cells such as insect cells (e.g. Drosophila such as Drosophila melanogaster), yeast cells, nematodes (such as Caenorhabditis elegans ( C. elegans )), mouse (such as Mus musculus) or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells and small murine erythroleukemia (MEL) cells) or human cells or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Suitably, the host cell is a mammalian cell, such as a human cell or an insect cell. In some embodiments, suitable host cells include E. coli.

Other suitable host cells that may generally be used in embodiments of the invention are those mentioned in the Examples section. Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of well-recognized techniques for introducing foreign nucleic acid molecules (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-glucose Glycan-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art.

When creating cell lines, it is generally preferred to produce stable cell lines. For example, for stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome depending on the expression vector and transfection technique used. To identify and select for these integrants, a gene encoding a selectable marker (eg, resistance to antibiotics) is typically introduced into the host cell along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin or methotrexate. A nucleic acid molecule encoding a selectable marker can be introduced into the host cell on the same vector, or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (eg, cells that have incorporated the selectable marker gene will survive while other cells die).

In one embodiment, a construct described herein is integrated into the genome of a host cell. The advantage of stable integration is that uniformity between individual cells or clones is achieved. Another advantage is that selection of the best producer can be performed. Therefore, it is desirable to create stable cell lines. In another embodiment, a construct described herein is transfected into a host cell. An advantage of transfecting the constructs into host cells is that protein production can be maximized. In one aspect, a cell comprising a nucleic acid construct or vector described herein is described. treatment method

In one aspect, provided herein is a method of treating skin cancer in a subject in need thereof, comprising administering to said subject (a) an IL-2 conjugate as described herein, and (b) a Western mipilimumab. In one aspect, provided herein is a method of treating skin cancer in a subject in need thereof, comprising administering to said subject (a) about 8 μg/kg, 16 μg/kg, or 24 μg/kg of An IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the method of treating skin cancer in a subject in need thereof comprises administering to said subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and ( b) Simiprimumab. In some embodiments, the method of treating skin cancer in a subject in need thereof comprises administering to said subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and ( b) Simiprimumab. In some embodiments, the method of treating skin cancer in a subject in need thereof comprises administering to said subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and ( b) Simiprimumab.

In another aspect, provided herein is an IL-2 conjugate for use in a method of treating skin cancer in a subject in need thereof, the method comprising administering to the subject (a) as described herein IL-2 conjugate of (b) simiprizumab. In another aspect, provided herein is an IL-2 conjugate for use in a method of treating skin cancer in a subject in need thereof, the method comprising administering to the subject (a) about 8 μg/ kg, 16 μg/kg or 24 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the methods comprise administering to the subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the methods comprise administering to the subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the methods comprise administering to the subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab.

In another aspect, provided herein is the use of an IL-2 conjugate in the manufacture of a medicament for use in a method of treating skin cancer in a subject in need thereof, the method comprising administering to the subject (a) An IL-2 conjugate as described herein, and (b) cimiprizumab. In another aspect, provided herein is the use of an IL-2 conjugate in the manufacture of a medicament for use in a method of treating skin cancer in a subject in need thereof, the method comprising administering to the subject (a) about 8 μg/kg, 16 μg/kg or 24 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the methods comprise administering to the subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the methods comprise administering to the subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the methods comprise administering to the subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab.

In yet another aspect, the methods described herein further comprise selecting a subject to be administered the IL-2 conjugate and cimiprizumab based at least in part on the skin cancer being unresectable skin cancer. In some aspects, the methods described herein further comprise selecting a subject to be administered the IL-2 conjugate and cimiprizumab based at least in part on the skin cancer being locally advanced cutaneous squamous cell carcinoma. In some aspects, the methods described herein further comprise selecting a subject to be administered the IL-2 conjugate and cimiprizumab based at least in part on the skin cancer being metastatic skin cancer. In some aspects, the methods described herein further comprise selecting a subject for administration of the IL-2 conjugate and cimiprizumab based at least in part on the skin cancer being unsuitable for topical therapy. In some aspects, the methods described herein further comprise selecting a subject to be administered the IL-2 conjugate and cimeprizumab based at least in part on the skin cancer being immune checkpoint inhibitor naïve .

In some aspects, provided herein are methods of treating skin cancer in a subject in need thereof, comprising selecting a subject with skin cancer, wherein the subject is selected based on one or more attributes, the attributes comprising (i) the skin cancer is unresectable skin cancer; (ii) the skin cancer is locally advanced squamous cell carcinoma of the skin; or (iii) the skin cancer is metastatic skin cancer; and The patient is administered (a) an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the methods comprise administering to the subject (a) about 8 μg/kg, 16 μg/kg, or 24 μg/kg of an IL-2 conjugate as described herein, and ( b) Simiprimumab. In some embodiments, the methods comprise administering to the subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the methods comprise administering to the subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the methods comprise administering to the subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab.

In some aspects, provided herein is the use of an IL-2 conjugate for stimulating CD8+ and/or NK cells in a subject in need thereof, the method comprising administering to the subject (a) as described herein The IL-2 conjugate described above, and (b) cimiprizumab. In some aspects, provided herein is the use of an IL-2 conjugate for stimulating CD8+ and/or NK cells in a subject in need thereof, the method comprising administering to the subject (a) about 8 μg /kg, 16 μg/kg or 24 μg/kg of IL-2 conjugates as described herein, and (b) cimiprizumab. In some embodiments, the method comprises administering to the subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the method comprises administering to the subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. In some embodiments, the method comprises administering to the subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and (b) cimiprizumab. cancer type

In some embodiments, the skin cancer is squamous cell skin cancer. In some embodiments, the skin cancer is squamous cell carcinoma of the skin. In some embodiments, the skin cancer is unresectable skin cancer, locally advanced cutaneous squamous cell carcinoma, or metastatic skin cancer. In some embodiments, the skin cancer is unresectable skin cancer. In some embodiments, the skin cancer is locally advanced squamous cell carcinoma of the skin. In some embodiments, the skin cancer is metastatic skin cancer. In some embodiments, the skin cancer is melanoma. In some embodiments, the skin cancer is basal cell carcinoma.

In some embodiments, the skin cancer is metastatic cutaneous squamous cell carcinoma (mCSCC) or locally advanced cutaneous squamous cell carcinoma (laCSCC) in a subject who is not undergoing curative surgery or curative candidate for radiation. In some embodiments, the skin cancer is metastatic cutaneous squamous cell carcinoma (mCSCC) in a subject who is not a candidate for curative surgery or curative radiation. In some embodiments, the skin cancer is locally advanced cutaneous squamous cell carcinoma (laCSCC) in a subject who is not a candidate for curative surgery or curative radiation. In some embodiments, the skin cancer is immune checkpoint inhibitor (ICI) naive metastatic cutaneous squamous cell carcinoma (CSCC).

In some embodiments, the skin cancer is refractory skin cancer. In some embodiments, the skin cancer is recurrent skin cancer. In some embodiments, the skin cancer is unresectable. In some embodiments, the skin cancer is metastatic. In some embodiments, the skin cancer is not amenable to topical therapy. In some embodiments, the skin cancer is advanced. In some embodiments, the skin cancer is immune checkpoint inhibitor (ICI) naïve. cast

In some embodiments, the subject is administered by intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intraarterial, intraarticular, intradermal, intravitreal, intraosseous infusion, intraperitoneal or intrathecal administration The patient was administered the IL-2 conjugate. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous, subcutaneous or intramuscular administration. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous administration. In some embodiments, the IL-2 conjugate is administered to the subject by subcutaneous administration. In some embodiments, the IL-2 conjugate is administered to the subject by intramuscular administration. In some embodiments, the IL-2 conjugate and cimiprizumab are administered to the subject by intravenous administration.

The IL-2 conjugate may be administered more than once, eg, two, three, four, five or more times. In some embodiments, the duration of treatment is up to 24 months, such as 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months months or 24 months. In some embodiments, the duration of treatment is further extended for up to an additional 24 months.

In some embodiments, the IL-2 conjugate is administered to the subject separately from the administration of cimiprizumab. In some embodiments, the IL-2 conjugate and cimiprizumab are administered to the subject sequentially. In some embodiments, the IL-2 conjugate is administered to the subject prior to administration of cimiprizumab to the subject. In some embodiments, the IL-2 conjugate is administered to the subject after cimiprizumab is administered to the subject. In some embodiments, the IL-2 conjugate and cimiprizumab are administered to the subject simultaneously.

In some embodiments, the IL-2 conjugate is administered to a subject in need thereof about once every two weeks, about once every three weeks, or about once every four weeks. In some embodiments, the IL-2 conjugate is administered biweekly to a subject in need thereof. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof once every three weeks. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof every 4 weeks. In some embodiments, the IL-2 conjugate is administered about every 14, 15, 16, 17, 18, 19, 20, or 21 days.

In some embodiments, cimiprizumab is administered to a subject in need thereof about every two weeks, about every three weeks, or about every four weeks. In some embodiments, cimiprizumab is administered biweekly to a subject in need thereof. In some embodiments, cimiprizumab is administered to a subject in need thereof every three weeks. In some embodiments, cimiprizumab is administered to a subject in need thereof every 4 weeks. In some embodiments, cimiprizumab is administered about every 14, 15, 16, 17, 18, 19, 20, or 21 days.

In some embodiments, the IL-2 conjugate and cimiprizumab are administered to a subject in need thereof about once every two weeks, about once every three weeks, or about once every four weeks. In some embodiments, the IL-2 conjugate and cimiprizumab are administered to a subject in need thereof biweekly. In some embodiments, the IL-2 conjugate and cimiprizumab are administered to a subject in need thereof once every three weeks. In some embodiments, the IL-2 conjugate and cimiprizumab are administered to a subject in need thereof once every 4 weeks. In some embodiments, the IL-2 conjugate and cimiprizumab are administered about every 14, 15, 16, 17, 18, 19, 20, or 21 days.

In some cases, the desired dose may conveniently be presented in a single dose or as divided doses either simultaneously (or within a short period of time) or at appropriate intervals (e.g. two, three, four one or more subdoses).

In some embodiments, cimiprizumab is administered every 3 weeks at a dose of about 350 mg. subjects

In some embodiments, the administration of the IL-2 conjugate and cimiprizumab is to an adult. In some embodiments, the adult is male. In other embodiments, the adult is female. In some embodiments, the subject is 18 years or older. In some embodiments, the adult is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old. In some embodiments, the administration of the IL-2 conjugate and simiprizumab is to an infant, child or adolescent. In some embodiments, the subject is at least 1 month, 2 months, 3 months, 6 months, 9 months, or 12 months old. In some embodiments, the subject is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 years old.

In some embodiments, the subject is a non-pregnant or non-lactating female. In some embodiments, the subject is a female who is not planning to have children. In some embodiments, the subject is a female of childbearing potential who is using an effective method of contraception for at least 180 days after discontinuation of treatment. In some embodiments, the subject is a male who does not donate sperm, abstain from heterosexual intercourse, or use effective contraception during treatment and for at least 210 days after discontinuation of treatment.

In some embodiments, the subject does not have an Eastern Cooperative Oncology Group (ECOG) performance status > 2. In some embodiments, the subject does not have a central nervous system (CNS) disease or a leptomeningeal disease. In some embodiments, the subject has no history of allogeneic or solid organ transplantation.

In some embodiments, the subject has not been previously treated with an immune checkpoint inhibitor except in the context of adjuvant or neoadjuvant drugs. In some embodiments, the subject has not received adjuvant or neoadjuvant therapy during the 6 months prior to developing skin cancer. In some embodiments, the subject is under antihypertensive treatment and is temporarily (for 12 to 48 hours) off antihypertensive medication prior to treatment.

In some embodiments, the subject has not received prior systemic therapy for advanced/metastatic disease (ie, skin cancer). In some embodiments, the subject has not received more than 2 prior lines of systemic therapy for advanced/metastatic disease (ie, skin cancer).

In some embodiments, the subject may undergo contrast-enhanced radiation response assessments before, during, and after treatment.

In some embodiments, the subject has a measurable disease (ie, skin cancer) as determined by RECIST v1.1. In some embodiments, the subject has at least one measurable lesion according to RECIST v1.1. In some embodiments, the subject has been determined to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. In some embodiments, the subject has adequate cardiovascular, hematological, hepatic, renal function and laboratory parameters, as determined by a physician. In some embodiments, the subject has been determined (eg, by a physician) to have a life expectancy of greater than or equal to 12 weeks. In some embodiments, the subject has been on prior anticancer therapy prior to the administration of the first therapeutic dose.

In some embodiments, the subject has squamous cell skin cancer. In some embodiments, the subject has squamous cell carcinoma of the skin. In some embodiments, the subject has unresectable skin cancer, locally advanced cutaneous squamous cell carcinoma, or metastatic skin cancer. In some embodiments, the subject has unresectable skin cancer. In some embodiments, the subject has locally advanced squamous cell carcinoma of the skin. In some embodiments, the subject has metastatic skin cancer. In some embodiments, the subject has melanoma. In some embodiments, the subject has basal cell carcinoma.

In some embodiments, the subject has metastatic cutaneous squamous cell carcinoma (mCSCC) or locally advanced cutaneous squamous cell carcinoma (laCSCC) and is not a candidate for curative surgery or curative radiation. In some embodiments, the subject has metastatic cutaneous squamous cell carcinoma (mCSCC) and is not a candidate for curative surgery or curative radiation. In some embodiments, the subject has locally advanced cutaneous squamous cell carcinoma (laCSCC) and is not a candidate for curative surgery or curative radiation. In some embodiments, the subject has received no more than 2 prior lines of systemic therapy. In some embodiments, the subject has immune checkpoint inhibitor (ICI) naïve metastatic cutaneous squamous cell carcinoma (CSCC).

In some embodiments, the subject has refractory skin cancer. In some embodiments, the subject has recurrent skin cancer. In some embodiments, the subject has unresectable skin cancer. In some embodiments, the subject has skin cancer that is not amenable to topical therapy. In some embodiments, the subject has advanced skin cancer. In some embodiments, the subject has immune checkpoint inhibitor (ICI) naïve skin cancer.

In some embodiments, the subject is immune checkpoint inhibitor (ICI) naive, has locally advanced, unresectable, or metastatic melanoma, and has not received prior therapy (i.e., IL-2 Conjugate therapy was 1L or first-line therapy; subjects were treatment naïve). That is, the subject will receive the IL-2 conjugate treatment as IL or first-line therapy. In some embodiments, the subject is a 1L melanoma subject. In some embodiments, the subject is a treatment naive melanoma subject. In some embodiments, the subject has a histologically confirmed diagnosis of unresectable locally advanced or metastatic melanoma not amenable to local therapy. In some embodiments, the subject does not have uveal melanoma or ocular melanoma or desmoplastic melanoma. In some embodiments, the subject has not received prior systemic therapy for advanced/metastatic skin cancer. In some embodiments, the subject has not received live virus vaccination within 28 days of starting IL-2 conjugate treatment.

In some embodiments, the subject is immune checkpoint inhibitor (ICI) naïve, has metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC, and is not curative surgery or curative radiation Candidate and have received no more than 2 prior lines of systemic therapy. That is, the subject will receive IL-2 conjugate therapy as a 1L, 2L or 3L therapy. In some embodiments, the subject is a 1L, 2L or 3L metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC subject. In some embodiments, the subject is a 1L CSCC subject. In some embodiments, the subject is a treatment naive CSCC subject. In some embodiments, the subject is a 2L CSCC subject. In some embodiments, the subject is a 3L CSCC subject. In some embodiments, the subject has a histologically confirmed diagnosis of locally advanced or metastatic cutaneous squamous cell carcinoma (CSCC). In some embodiments, the subject does not have vermilion (vermilion) or anogenital region as the primary site of CSCC and mixed CSCC histology (eg, sarcomatoid, adenosquamous). In some embodiments, the subject has not received more than 2 prior lines of systemic therapy for advanced/metastatic skin cancer.

In some embodiments, the subject has an ECOG performance status of less than 2. In some embodiments, the subject has no history of allogeneic tissue or solid organ transplantation. In some embodiments, the subject has no immune-mediated/related toxicities from or resulting in interruption of prior tier 4 immuno-oncology therapy. In some embodiments, the subject does not have an ongoing AE of Grade > 2 resulting from any prior anticancer therapy. In some embodiments, the subject does not have a baseline oxygen saturation (Sp02) of < 92% (without oxygen therapy). In some embodiments, the subject does not have active brain metastases or leptomeningeal disease. In some embodiments, the subject does not have a lung disease. In some embodiments, the subject does not have comorbidities requiring corticosteroid therapy. In some embodiments, the subject may be temporarily (at least 36 hours) off antihypertensive medication prior to each IL-2 conjugate administration. In some embodiments, the subject is free of any medical or clinical condition, laboratory abnormality, or any particular condition that, in the judgment of the supervising physician, may preclude protocol therapy or may render the subject unsuitable for the study. In some embodiments, the subject has not received antibiotics (excluding topical antibiotics) within 14 days of administration of the first dose of IL-2 conjugate. In some embodiments, the subject has not had severe or unstable cardiac disease within 6 months of starting IL-2 conjugate treatment. In some embodiments, the subject has not had an active, known or suspected autoimmune disease requiring systemic therapy within 2 years of starting IL-2 conjugate therapy. In some embodiments, the subject does not have a known second malignancy that has progressed or required active treatment within 3 years of starting IL-2 conjugate therapy.

In some embodiments, a subject with skin cancer is selected for treatment based on one or more attributes including (i) the skin cancer is an unresectable skin cancer; (ii) the skin cancer is locally advanced squamous cell carcinoma of the skin; or (iii) the skin cancer is metastatic skin cancer. In some embodiments, a subject with skin cancer is selected for treatment based on (i) the skin cancer is unresectable skin cancer. In some embodiments, the subject with skin cancer is selected for treatment based on (ii) the skin cancer is locally advanced cutaneous squamous cell carcinoma. In some embodiments, the subject with skin cancer is selected for treatment based on (iii) the skin cancer is metastatic skin cancer. In some embodiments, a subject with skin cancer is selected for treatment based on (i) the skin cancer is unresectable skin cancer, and (ii) the skin cancer is locally advanced cutaneous squamous cell carcinoma. In some embodiments, a subject with skin cancer is selected for treatment based on (i) the skin cancer is unresectable skin cancer, and (iii) the skin cancer is metastatic skin cancer. In some embodiments, a subject with skin cancer is selected for treatment based on (ii) the skin cancer is locally advanced cutaneous squamous cell carcinoma, and (iii) the skin cancer is metastatic skin cancer. In some embodiments, a patient with skin cancer is selected based on (i) the skin cancer is unresectable skin cancer, (ii) the skin cancer is locally advanced cutaneous squamous cell carcinoma, and (iii) the skin cancer is metastatic skin cancer. Subjects are used for treatment.

In some embodiments, a subject with skin cancer is selected for treatment based at least in part on the skin cancer being unresectable skin cancer. In some embodiments, a subject with skin cancer is selected for treatment based at least in part on the skin cancer being locally advanced cutaneous squamous cell carcinoma. In some embodiments, a subject with skin cancer is selected for treatment based at least in part on the skin cancer being metastatic skin cancer. In some embodiments, a subject with skin cancer is selected for treatment based at least in part on the skin cancer being unsuitable for topical therapy. In some embodiments, a subject having skin cancer is selected for treatment based at least in part on the skin cancer being immune checkpoint inhibitor naïve.

In some embodiments, the subject has no known hypersensitivity or contraindications to any IL-2 conjugate, PEG, pegylated drug, or cimiprizumab disclosed herein. cast effect

In some embodiments, the administration of the IL-2 conjugate and cimiprizumab provides a complete response, partial response, or stable disease. In some embodiments, administration of the IL-2 conjugate and cimiprizumab provides a complete response. In some embodiments, administration of the IL-2 conjugate and cimiprizumab provides a partial response. In some embodiments, the administration of the IL-2 conjugate and cimiprizumab provides disease stabilization.

In some embodiments, the administration of the IL-2 conjugate and cimiprizumab reduces target lesion size. In some embodiments, the administration of the IL-2 conjugate and cimiprizumab stabilizes target lesion size. In some variations, the administration of the IL-2 conjugate and cimiprizumab slows the growth rate of the target lesion. In some variations, the administration of the IL-2 conjugate and cimiprizumab arrests the growth of the target lesion. In some embodiments, the administration of the IL-2 conjugate and cimiprizumab eliminates the target lesion.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause vascular leak syndrome in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause Grade 2, Grade 3, or Grade 4 vascular leak syndrome in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause Grade 2 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate and cimiprizumab to the subject does not cause Grade 3 vascular leak syndrome in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause Grade 4 vascular leak syndrome in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not result in loss of vascular tone in the subject.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause extravasation of plasma proteins and fluids of the subject into the extravascular space.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause hypotension and decreased organ perfusion in the subject.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not result in impaired neutrophil function in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not result in decreased chemotaxis in the subject.

In some embodiments, administration of the IL-2 conjugate and cimiprizumab to the subject is not associated with an increased risk of disseminated infection in the subject. In some embodiments, the disseminated infection is sepsis or bacterial endocarditis. In some embodiments, the disseminated infection is sepsis. In some embodiments, the disseminated infection is bacterial endocarditis. In some embodiments, the subject is treated for any pre-existing bacterial infection prior to administering the IL-2 conjugate and cimiprizumab. In some embodiments, the subject is treated with an antibacterial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administering the IL-2 conjugate and cimiprizumab. tester.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not exacerbate pre-existing or initial manifestations of an autoimmune or inflammatory disorder in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not exacerbate pre-existing or initial manifestations of the autoimmune disease in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not exacerbate pre-existing or initial manifestations of the inflammatory disorder in the subject. In some embodiments, the subject's autoimmune disease or inflammatory disorder is selected from Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes, ocular myasthenia gravis, crescentic IgA nephropathy Glomerulonephritis, cholecystitis, cerebral vasculitis, Smith-Johnson syndrome, and bullous pemphigoid. In some embodiments, the subject's autoimmune or inflammatory disorder is Crohn's disease. In some embodiments, the subject's autoimmune or inflammatory disorder is scleroderma. In some embodiments, the subject's autoimmune or inflammatory disorder is thyroiditis. In some embodiments, the subject's autoimmune disease or inflammatory disorder is inflammatory arthritis. In some embodiments, the subject's autoimmune or inflammatory disorder is diabetes. In some embodiments, the subject's autoimmune or inflammatory disorder is ocular myasthenia gravis. In some embodiments, the subject's autoimmune disease or inflammatory disorder is crescentic IgA glomerulonephritis. In some embodiments, the subject's autoimmune or inflammatory disorder is cholecystitis. In some embodiments, the subject's autoimmune or inflammatory disorder is cerebral vasculitis. In some embodiments, the subject's autoimmune disease or inflammatory disorder is Smith-Johnson syndrome. In some embodiments, the subject's autoimmune or inflammatory disorder is bullous pemphigoid.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause changes in mental status, speech difficulties, cortical blindness, limb or gait ataxia in the subject , hallucinations, agitation, dullness, or coma. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause seizures in the subject. In some embodiments, administration of the IL-2 conjugate and cimiprizumab to a subject is not contraindicated in subjects with a known seizure disorder.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause capillary leak syndrome in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause Grade 2, Grade 3, or Grade 4 capillary leak syndrome in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause Grade 2 capillary leak syndrome in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause Grade 3 capillary leak syndrome in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause Grade 4 capillary leak syndrome in the subject.

In some embodiments, administration of the IL-2 conjugate and cimiprizumab to the subject does not result in a decrease in mean arterial blood pressure in the subject following administration. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject causes hypotension in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause the subject to experience a systolic blood pressure of less than 90 mm Hg or a drop from baseline systolic blood pressure of 20 mm Hg.

In some embodiments, administration of the IL-2 conjugate and cimiprizumab to the subject does not cause edema or impairment of renal or hepatic function in the subject.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not cause eosinophilia in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not result in an eosinophil count in the peripheral blood of the subject exceeding 500/μL. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not result in an eosinophil count in the peripheral blood of the subject exceeding 500/µL to 1500/µL . In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not result in an eosinophil count in the peripheral blood of the subject exceeding 1500/μL to 5000/μL . In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not result in an eosinophil count in the peripheral blood of the subject exceeding 5000/μL. In some embodiments, administration of the IL-2 conjugate and cimiprizumab to a subject is not contraindicated in subjects receiving an existing psychotropic drug regimen.

In some embodiments, administration of the IL-2 conjugate and cimiprizumab to a subject is not contraindicated in subjects receiving existing regimens of nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drugs of. In some embodiments, the IL-2 conjugate and cimiprizumab are administered to a subject in the presence of aminoglycosides, cytotoxic chemotherapy, doxorubicin, methotrexate, or asparagine. Aminidase is not contraindicated in subjects on existing regimens. In some embodiments, administration of the IL-2 conjugate and cimiprizumab to a subject is not contraindicated in subjects receiving a combination regimen containing an antineoplastic agent. In some embodiments, the antineoplastic agent is selected from dacarbazine, cisplatin, tamoxifen, and interferon-alpha.

In some embodiments, administration of the IL-2 conjugate and cimiprizumab to the subject does not result in one or more grade 4 adverse events in the subject following administration. In some embodiments, the grade 4 adverse event is selected from the group consisting of hypothermia; shock; bradycardia; premature ventricular contraction; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; stagnation; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; abnormal liver function tests; gastrointestinal bleeding; hematemesis; bloody diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis ; anemia; leukopenia; leukocytosis; hypocalcemia; elevated alkaline phosphatase; elevated blood urea nitrogen (BUN); hyperuricemia; elevated nonprotein nitrogen (NPN); respiratory acidosis; lethargy ; agitation; neuropathy; paranoid reactions; convulsions; epileptic grand mal convulsions; delirium; asthma, pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; failure; and acute tubular necrosis. In some embodiments, administration of the IL-2 conjugate and cimiprizumab to the subject does not result in one or more grade 4 adverse events in greater than 1% of the subjects following administration. In some embodiments, the grade 4 adverse event is selected from the group consisting of hypothermia; shock; bradycardia; premature ventricular contraction; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; stagnation; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; abnormal liver function tests; gastrointestinal bleeding; hematemesis; bloody diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis ; anemia; leukopenia; leukocytosis; hypocalcemia; elevated alkaline phosphatase; elevated blood urea nitrogen (BUN); hyperuricemia; elevated nonprotein nitrogen (NPN); respiratory acidosis; lethargy ; agitation; neuropathy; paranoid reactions; convulsions; epileptic grand mal convulsions; delirium; asthma, pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; failure; and acute tubular necrosis.

In some embodiments, administration of the IL-2 conjugate and cimiprizumab to a group of subjects does not cause one or more adverse events in more than 1% of the subjects following administration, wherein all The one or more adverse events are selected from the group consisting of duodenal ulceration; intestinal necrosis; myocarditis; supraventricular tachycardia; permanent or temporary blindness secondary to optic neuritis; transient ischemic attack; meningitis; cerebral edema; pericardium inflammation; allergic interstitial nephritis; and tracheoesophageal fistula.

In some embodiments, administration of the IL-2 conjugate and cimiprizumab to a group of subjects does not cause one or more adverse events in more than 1% of the subjects following administration, wherein all The one or more adverse events are selected from malignant hyperthermia; cardiac arrest; myocardial infarction; pulmonary embolism; stroke; intestinal perforation; liver or kidney failure; major depression leading to suicide; pulmonary edema; respiratory arrest; respiratory failure.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject stimulates CD8+ cells in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject stimulates NK cells in the subject. Stimulation can include an increase in the number of CD8+ cells in the subject, eg, about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration. In some embodiments, the CD8+ cells comprise memory CD8+ cells. In some embodiments, the CD8+ cells include effector CD8+ cells. Stimulation can include, for example, an increase in the proportion of Ki67-positive CD8+ cells in the subject about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration. Stimulation can include, for example, an increase in the number of NK cells in a subject about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject expands the number of CD8+ cells in the subject. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject expands the number of NK cells in the subject.

In some embodiments, the eosinophils expand in the subject by no more than about 2-fold, such as no more than about 1.5-fold, 1.4-fold, after administration of the IL-2 conjugate and cimiprizumab or 1.3 times. In some embodiments, the CD4+ cells expand in the subject by no more than about 2-fold, such as by no more than about 1.8-fold, 1.7-fold, or 1.6-fold following administration of the IL-2 conjugate and cimiprizumab. times. In some embodiments, CD8+ cells and/or NK cells expand more than CD4+ cells and/or eosinophils in the subject following administration of the IL-2 conjugate and simiprizumab expansion. In some embodiments, CD8+ cells are expanded more than CD4+ cells. In some embodiments, NK cells are expanded more than CD4+ cells. In some embodiments, CD8+ cells are expanded more than eosinophils. In some embodiments, NK cells are expanded more than eosinophils. Fold amplification was determined relative to the baseline value measured prior to administration of the IL-2 conjugate. In some embodiments, the fold expansion is determined at any time after administration, such as about 4, 5, 6 or 7 days after administration, or about 1, 2, 3 or 4 weeks after administration.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to a subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of CD8+ T and NK cells in the subject The number of peripheral CD4+ regulatory T cells. In some embodiments, administering the IL-2 conjugate and cimiprizumab to a subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of CD8+ T and NK cells in the subject The number of peripheral eosinophils. In some embodiments, administering the IL-2 conjugate and cimiprizumab to a subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of CD8+ T and NK cells in the subject The number of CD8+ T cells and NK cells in the tumor without increasing the number of CD4+ regulatory T cells in the tumor in the subject.

In some embodiments, administering the IL-2 conjugate and cimiprizumab to a subject does not require the use of an intensive care facility or a skilled cardiopulmonary or intensive care medical specialist. In some embodiments, administering the IL-2 conjugate and cimiprizumab to a subject does not require the use of an intensive care facility or a skilled cardiopulmonary or intensive care medical specialist. In some embodiments, administering the IL-2 conjugate and cimiprizumab to a subject does not require the use of an intensive care facility. In some embodiments, administering the IL-2 conjugate and cimiprizumab to the subject does not require the use of a skilled cardiopulmonary or critical care medical specialist.

In some embodiments, administration of the IL-2 conjugate and cimiprizumab does not cause dose limiting toxicity. In some embodiments, administration of the IL-2 conjugate and cimiprizumab does not cause severe cytokine release syndrome. In some embodiments, the IL-2 conjugate does not induce anti-drug antibodies (ADA), ie, antibodies raised against the IL-2 conjugate. In some embodiments, ADA-induced deficiency is determined by direct immunoassay with anti-PEG antibody and/or ELISA with anti-IL-2 conjugate antibody. An IL-2 conjugate was not considered to induce ADA if the measured ADA levels were statistically indistinguishable from baseline (pre-treatment) levels or from untreated controls. additional medicine

In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents include one or more analgesics. In some embodiments, the one or more analgesics include acetaminophen. In some embodiments, the one or more additional therapeutic agents include one or more antihistamines. In some embodiments, the one or more antihistamines include diphenhydramine. In some embodiments, the one or more additional therapeutic agents include one or more serotonin 5-HT ₃ receptor antagonists. In some embodiments, the one or more serotonin 5-HT ₃ receptor antagonists comprises ondansetron. In some embodiments, one or more additional therapeutic agents, such as acetaminophen, diphenhydramine, and/or ondansetron, are administered to the subject prior to administration of the IL-2 conjugate. In some embodiments, one or more additional therapeutic agents, such as acetaminophen, diphenhydramine, and/or ondansetron, are administered to the subject following administration of the IL-2 conjugate. In some embodiments, one or more additional therapeutic agents, such as acetaminophen, diphenhydramine, and/or ondansetron, are administered to the subject concurrently with the administration of the IL-2 conjugate. Set / Product

In certain embodiments, disclosed herein are kits and articles of manufacture for use with one or more of the methods and compositions described herein. Such kits include carriers, packages, or containers that are compartmentalized to house one or more containers, such as vials, tubes, etc., each of the one or more containers containing an individual drug to be used in the methods described herein. one of the elements. Suitable containers include, for example, bottles, vials, syringes and test tubes. In one embodiment, the container is formed from various materials such as glass or plastic.

Kits typically include a label listing the contents and/or directions for use, and a package insert containing the directions for use. Usually a set of instructions will also be included.

In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers or other characters forming the label are affixed, molded or etched into the container itself; A label is attached to the container, for example as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents as in the methods described herein.

In certain embodiments, pharmaceutical compositions are presented in a pack or dispenser device containing one or more unit dosage forms containing a compound provided herein. Packagings, for example, contain metal or plastic foils, such as blister packs. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the package or dispenser is also accompanied by a notice associated with the container in a form prescribed by the governmental agency regulating the manufacture, use or sale of pharmaceuticals, the notice reflecting the agency's Approval of drug forms for veterinary administration. Examples of such notices are FDA-approved drug labels, or approved product inserts. In one embodiment, a composition containing a compound provided herein formulated in a compatible pharmaceutical carrier is also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. example

These examples are provided for illustrative purposes only, and do not limit the scope of the claims presented herein. Example 1. Preparation of pegylated IL-2 conjugates.

Exemplary methods for preparing the IL-2 conjugates described herein are provided in detail in this Example.

流程 2.

實例 2. 投予 IL-2 接合物（ 8 μg/kg 、 16 μg/kg 和 24 μg/kg [Q3W] ）後生物標記物影響的臨床研究。   使用 24 μg/kg 劑量 [Q3W] 的群組 IL-2 for bioconjugation was expressed as inclusion bodies in E. coli using the methods disclosed herein using: (a) expression of a plastid encoding (i) a protein with the desired amino acid sequence whose gene contains The first unnatural base pair to provide a codon at the desired position for incorporation of the unnatural amino acid N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) , and (ii) a tRNA derived from M. mazei Pyl whose gene contains a second unnatural nucleotide to provide a matching anticodon in place of its native sequence; (b) encoding a M. Plastids of pyrrolysyl-tRNA synthetase ( Mb PylRS), (c) N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK); and (d ) A truncated variant of the nucleotide triphosphate transporter PtNTT2 in which the first 65 amino acid residues of the full-length protein are missing. The double-stranded oligonucleotide encoding the amino acid sequence of the desired IL-2 variant contains the codon AXC as codon 64 of the sequence encoding the protein having SEQ ID NO: 1, wherein P64 is replaced by the non-natural Amino acid substitution. The plastid encoding the orthogonal tRNA gene from M. mazei contained the AXC-matched anticodon GYT in place of its native sequence, where Y is an unnatural nucleotide disclosed herein. X and Y are selected from the non-natural nucleotides dTPT3 and dNaM disclosed herein. Expressed proteins were extracted from inclusion bodies and refolded using standard procedures, followed by site-specific PEGylation of the AzK-containing IL-2 product using DBCO-mediated copper-free click chemistry to incorporate stable covalent The mPEG moiety is attached to AzK. Exemplary reactions are shown in Schemes 1 and 2 (where n represents the number of repeating PEG units). Reaction of the AzK moiety with the alkynyl moiety of DBCO may provide a regioisomeric product or a mixture of regioisomeric products. Process 1 .

Process 2.

Example 2. Clinical study of biomarker effects following administration of IL-2 conjugates ( 8 μg/kg , 16 μg/kg and 24 μg/kg [Q3W] ).   Cohort using 24 μg/kg dose [Q3W]

Studies were performed to characterize the immunological effects of in vivo administration of the IL-2 conjugates described herein. The IL-2 conjugate comprises SEQ ID NO: 2, wherein position 64 is AzK_L1_PEG30kD, wherein AzK_L1_PEG30kD is defined as having the following structure: formula (IV) or formula (V) or formula (IV) and formula (V) mixture, and a 30 kDa linear mPEG chain. The IL-2 conjugate may also be described as comprising an IL-2 conjugate of SEQ ID NO: 1, wherein position 64 is replaced to have the following structure: Formula (IV) or Formula (V) or Formula (IV) and A mixture of formula (V), and a 30 kDa linear mPEG chain. The IL-2 conjugate can also be described as comprising an IL-2 conjugate of SEQ ID NO: 1, wherein position 64 is replaced to have the following structure: Formula (XII) or Formula (XIII) or Formula (XII) and A mixture of formula (XIII), and a 30 kDa linear mPEG chain. Compounds were prepared using a method in which a protein having SEQ ID NO: 1 was first prepared, wherein the proline at position 64 was replaced by N 6-((2-azidoethoxy)-carbonyl)-L-lysine AzK . The AzK-containing protein was then allowed to react under click chemistry conditions with DBCO containing a methoxyl linear PEG group with an average molecular weight of 30 kDa, followed by purification and formulation using standard procedures. In the cohort here and throughout Example 2, the mass of drug per kg of subject (eg, 24 μg/kg) refers to the mass of IL-2 excluding the mass of PEG and linker .

IL-2 conjugates were administered every 3 weeks [Q3W] at a dose of 24 μg/kg by IV infusion over 30 minutes. Analysis of the effect on the following biomarkers as surrogate predictors of safety and/or efficacy: Eosinophilia (elevated peripheral eosinophil count): IL-2 associated with vascular leak syndrome (VLS) Cell replacement marker of induced cell (eosinophil) proliferation; Interleukin 5 ( IL-5 ) : IL-2-induced priming of type 2 innate lymphocytes and leading to hypereosinophilia and potential VLS Interleukin 6 ( IL-6 ) : A cytokine replacement marker for IL-2-induced cytokine release syndrome (CRS); and Interferon gamma ( IFN-γ ): Cytokine replacement marker of IL-2-induced CD8+ cytotoxic T lymphocyte priming.

The effect on the following biomarkers was analyzed as a surrogate predictor of anti-tumor immune activity: Peripheral CD8+ effector cells: a marker of IL-2-induced proliferation of these target cells in the periphery, after infiltration a surrogate for induction of a possible potential therapeutic response Markers; Peripheral CD8+ memory cells: a marker of IL-2-induced proliferation of these target cells in the periphery, after infiltration a surrogate marker for induction of potentially therapeutic and maintenance memory populations that may persist; peripheral NK cells: IL-2 Induced markers of proliferation of these target cells in the periphery, after infiltration become surrogate markers for induction of potentially rapid therapeutic responses; and Peripheral CD4+ regulatory cells: markers of IL-2-induced proliferation of these target cells in the periphery, in Infiltration becomes a surrogate marker for induction of immunosuppressive TME and counteracting effector-based therapeutics.

Subjects were male or female aged ≥ 18 years at screening. All subjects have been previously treated with anticancer therapy and meet at least one of the following: treatment-related toxicity resolved to grade 0 or 1 according to NCI CTCAE v5.0 (except alopecia); or treatment-related toxicity according to NCI CTCAE v5.0 Regression to at least grade 2, prior approval by medical monitor. The most common tumors are colorectal cancer or melanoma.

Subjects also met the following criteria: Provide informed consent. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Investigator-determined life expectancy greater than or equal to 12 weeks. Histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors. Advanced or metastatic solid tumors who refuse standard of care; or where no reasonable standard of care would confer clinical benefit; or who are intolerable, ineffective, or unavailable to standard therapy. Disease measurable according to RECIST v1.1. Appropriate laboratory parameters include: absolute lymphocyte count > 0.5 times the lower limit of normal; platelet count > 100 × 10 ⁹ /L; hemoglobin > 9.0 g/dL (without growth factors or transfusions within 2 weeks; ESA and CSF administered 1 week clearance is sufficient); absolute neutrophil count > 1.5 × 10 ⁹ /L (growth factor-free for 2 weeks); prothrombin time (PT) and partial thromboplastin time (PTT) ≤ 1.5 times upper limit of normal (ULN); aspartate transaminase (AST) and alanine transaminase (ALT) ≤ 2.5 times ULN, unless liver metastases may be ≤ 5 times ULN; total bilirubin ≤ 1.5 × ULN. Premenopausal women and women less than 12 months postmenopausal had a negative serum pregnancy test within 7 days before starting study treatment.

Q3W dosing. Eleven individuals (4 [36.4%] males, 7 [63.6%] females, 9 [81.8%] Caucasians) received a 24 μg/kg dose of IL-2 conjugate Q3W for up to nine cycles (1 dose per cycle). In the discussion of the first cohort here and throughout Example 2, the mass of drug per kg of subject (eg, 24 μg/kg) refers to the mass of IL-2 excluding the mass of PEG and linker .

One subject had a partial response at the initial scan, confirmed at the second and third scans (prior PD-1 exposure), for 6+ months; five subjects had initially stable disease (at 6 weeks assessment), three subjects had disease progression at the first assessment, and one subject withdrew from treatment due to an adverse event. The peak expression levels of CD8+ Ki67 in all subjects exceeded 50% (50%-85%) after administration.

A 73-year-old male subject with squamous cell carcinoma of unknown origin who received 7 cycles of treatment (24 µg/kg Q3W) and two systemic therapies including anti-PD-1 line (best response to anti-PD-1: SD), showing a 31% tumor reduction after two cycles. The largest tumor responses were found in patients with other immunosensitive tumors to be renal cell carcinoma (RCC) (16% growth) and melanoma (10% growth; 2% reduction; and 20% reduction observed in two subjects).

Peripheral expansion of CD8+ T effector cells averaged 4.47-fold higher than baseline. All subjects had elevated levels of NK cell Ki67 expression after administration. The peak peripheral expansion of NK cells of the subjects after administration was 7.67 times higher than the baseline on average.

Efficacy biomarkers. Efficacy biomarker data were based on available data from up to 10 subjects receiving 24 µg/kg IL-2 conjugate. Measure peripheral CD8+ _Teff cell counts ( Figure 1A -Figure 1C ). Prolonged CD8+ expansion over baseline (eg, greater than or equal to a 2-fold change) was observed in some subjects 3 weeks after prior dosing. The percentage of CD8+ T _eff cells expressing Ki67 was also measured ( Figure 2 ). Figures 3A - 3B show peripheral CD8+ memory cell counts.

Figures 4A - 4D show peripheral NK cell counts. An increase in NK cell counts was observed in each subject. The percentage of NK cells expressing Ki67 was also measured ( Figure 5 ).

Figures 6A - 6B show peripheral CD4+ T _reg counts. The percentage of CD4+ T _reg cells expressing Ki67 was also measured ( FIG. 7 ).

Measure eosinophil counts ( Figure 8A- Figure 8C ). As reported in Pisani et al., Blood 1991 Sep 15;78(6):1538-44, in patients with IL-2-induced hypereosinophilia, the measured increase did not exceed four times and always below the range of 2328-15958 eosinophils/μL. Levels of IFN-γ, IL-5 and IL-6 were also measured ( FIG. 9A- FIG. 9C ). Measurements showed induction of IFN-γ but less IL-5 and IL-6 (cytokines associated with VLS and CRS, respectively), except for one subject whose IL-6 levels were lower after treatment (receiving zizumab) increased to approximately 1100 pg/mL at 24 hours, but declined thereafter.

Anti-drug antibodies ( ADA ). Anti-drug antibodies (ADA) were assayed in samples from treated subjects after each dosing period. Anti-PEG autoantibodies were detected by direct immunoassay (limit of detection: 36 ng/mL). A bridged MesoScale Discovery ELISA with a labeled form of the IL-2 conjugate had a detection limit of 4.66 ng/mL. Additionally, a cell-based assay for neutralizing antibodies against the IL-2 conjugate was performed using the CTLL-2 cell line with STAT5 phosphorylation as readout (limit of detection: 6.3 μg/mL).

Samples were collected and analyzed after each dose cycle from two subjects who received 5 dose cycles and one subject who received 4 dose cycles. Assay-specific cut-off points were determined during assay quantification with a signal to negative ratio of 1.09 or higher for the IL-2 conjugate ADA assay and 2.08 for the PEG ADA assay. Confirmatory testing was performed on samples that gave positive or indeterminate results in the IL-2 conjugate assay in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in blocking solution) Assay samples and controls. A confirmatory test was performed on samples giving positive or indeterminate results in the PEG assay, where samples were assayed in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in 6% horse serum) and contrast. Samples were considered "confirmed" if, during the detection step, the absorbance signal of the sample was suppressed by equal to or greater than the assay-specific cut-off point determined during assay quantification (14.5% for IL-2 conjugates, or 42.4% for PEG) . No confirmed ADA was detected against IL-2 conjugates or PEG (data not shown).

Summary of Results; Discussion. All tested subjects had post-dose CD8+ Ki67 expression levels greater than 50% (50%-85%) at one or more time points, and peripheral expansion of CD8+ T effector (Teff) cells. All tested subjects also had post-dose NK cell Ki67 expression levels greater than 50% (50%-100%) at one or more time points, as well as peripheral expansion of NK cells. IL-5 levels did not rise meaningfully, and subjects whose IL-6 levels rose on Day 3 showed a decrease the following day. ADA was not induced in any of the subjects tested.

An AE is any adverse medical event, regardless of cause, in a clinical research subject administered a drug product. Dose-limiting toxicity was defined as an AE occurring within ±1 day from Day 1 to Day 29 (inclusive) of the treatment cycle that was not clearly or indisputably related to an extrinsic cause only and that met at least one of the following criteria: 3 Grade 4 neutropenia (absolute neutrophil count < 1000/mm ³ > 500/mm ³ ) lasting ≥ 7 days, or Grade 4 neutropenia of any duration • Grade 3+ Febrile neutropenia • Grade 4 + thrombocytopenia (platelet count < 25,000/mm ³ ) • Grade 3 + thrombocytopenia (platelet count < 50,000-25,000/mm ³ ) for ≥ 5 days, or Associated with clinically significant bleeding or requiring platelet transfusion • Failure to meet recovery criteria within 10 days of an absolute neutrophil count of at least 1,000 cells/ ^mm3 and a platelet count of at least 75,000 cells/ ^mm3 • Any other classification 4+ hematological toxicity lasting ≥ 5 days • Grade 3+ ALT or AST and bilirubin >2 times ULN, no evidence of cholestasis or other causes such as viral infection or other drugs (i.e. Hy's law) • Premedication occurred Grade 3 Infusion-Related Reactions; Grade 4 Infusion-Related Reactions Grade 3 Vascular Leak Syndrome, defined as hypotension associated with fluid retention and pulmonary edema Grade 3+ Anaphylaxis Grade 3+ Hypotension Grade 3+AEs Does not resolve to <Grade 2 within 7 days of starting standard of care medical management • Grade 3 + cytokine release syndrome The following exceptions apply for non-hematologic AEs: • Grade 3 fatigue, nausea, vomiting, or diarrhea within ≤ 3 days Resolution by best medical management to ≤ Grade 2 • Grade 3 fever (defined as >40ºC for ≤ 24 hours) • Grade 3 infusion-related reaction, occurred in the absence of premedication; subsequent doses should use premedication, AND if response recurs, DLT Grade 3 arthralgia or rash that resolves to ≤ Grade 2 within 7 days of starting standard-of-care medical management (eg, systemic corticosteroid therapy) if subject was Grade 1 or For grade 2 ALT or AST elevation (considered to be indirectly caused by liver metastasis), the grade 3 elevation must also be ≥ 3 times baseline and last for > 7 days.

A serious AE was defined as any AE leading to any of the following outcomes: death; life-threatening AE; hospitalization or prolongation of current hospitalization; persistent or severe loss or severe impairment of ability to perform normal life functions; Anomalies/Birth Defects. A medically important event that is likely not to result in death, is life-threatening, or requires hospitalization may be considered serious when, based on appropriate medical judgment, it may endanger the subject and may require medical or surgical intervention to prevent one of the outcomes listed above event. Examples of such medical events include allergic bronchospasm requiring intensive treatment in the emergency department or at home, blood dyscrasias or convulsions that do not result in hospitalization, or development of drug dependence or substance abuse.

No dose-limiting toxicities were reported. No cumulative toxicity. There were two treatment-related SAEs (1 G3 acute kidney injury and 1 G4 cytokine release syndrome), which resolved with accepted standard of care. In conclusion, IL-2 conjugates were considered to be well tolerated.

All subjects had at least one treatment-emergent AE (TEAE). The TEAEs are detailed in Table 1. There were no grade 5 TEAEs. Two subjects experienced Grade 3 events, and three subjects experienced Grade 4 events. Grade 3 events included: 1 ALT/AST increase, 1 neutrophil count decrease, and 1 acute kidney injury. Grade 4 events included: 1 CRS, 1 increase in lymphocyte count, and 2 decreases in lymphocyte count. Table 1 Treatment-emergent adverse events (TEAEs) (n=11) IL-2 conjugate 24 µg/kg Q3W (N=11) Major system organ class preference, n (%) all grades Grading≥3 _ Number of participants with TEAE 11 (100) 8 (72.7) Infection and Infestation 1 (9.1) 0 Blood and Lymphatic System Disorders 2 (18.2) 1 (9.1) immune system disorder 2 (18.2) 1 (9.1) Metabolic and Nutritional Disorders 4 (36.4) 0 mental disorder 1 (9.1) 0 nervous system disorder 3 (27.3) 0 heart disorder 2 (18.2) 0 Vascular disorders 3 (27.3) 1 (9.1) Respiratory, thoracic and mediastinal disorders 4 (36.4) 1 (9.1) Gastrointestinal disorders 4 (36.4) 0 Skin and Subcutaneous Tissue Disorders 2 (18.2) 0 Musculoskeletal and connective tissue disorders 1 (9.1) 0 Kidney and urinary system disorders 1 (9.1) 1 (9.1) Systemic disorders and administration site conditions 9 (81.8) 1 (9.1) an examination 9 (81.8) 6 (54.5) Injury, poisoning and surgical complications 0 0

TEAE mainly consists of flu-like symptoms, nausea or vomiting. TEAEs resolved with accepted standard of care. Treatment-related AEs were transient. AEs of fever, hypotension, and hypoxia were not associated with elevated IL-5/IL-6 cytokines. One subject experienced an increase in IL-6 to 1000 pg/mL at 24 hours (after tocilizumab treatment), which decreased to less than 100 pg/mL by 72 hours. No significant effect on vital signs, no QTc prolongation or other cardiotoxicity.

Thus, the IL-2 conjugate exhibited encouraging PD profiles and was generally well tolerated. The in vivo half-life of the IL-2 conjugate was determined to be about 10 hours. Taken together, these results are considered to support non-α-preferential activity of IL-2 conjugates, with a tolerable safety profile, encouraging PD and preliminary evidence of activity in patients with immunosensitive tumors.   Cohorts using 8 μg/kg and 16 μg/kg doses [Q3W ]

IL-2 conjugates were administered by IV infusion every 3 weeks [Q3W] at doses of 8 μg/kg or 16 μg/kg over 30 minutes. For the study using the 24 μg/kg dose, the effect on the same biomarkers described above was analyzed as a surrogate predictor of safety and/or efficacy. Subjects in these studies met the same criteria as subjects receiving the 24 μg/kg dose. Cohort 1 : Dosing at 8 μg/kg [Q3W] .

Cohort 1 (individuals with malignant solid tumors) received the IL-2 conjugate at a dose of 8 μg/kg Q3W for five dose cycles.

Four individuals with initially stable disease (at the 6-week assessment; one patient had ~12% tumor regression) were treated with the IL-2 conjugate. The post-dose CD8+ Ki67 expression levels of these four subjects exceeded 60% (65%-80%).

Biomarkers were determined for 4 individuals in Cohort 1 as follows. Peripheral expansion of CD8+ T effector cells averaged 1.53-fold above baseline; one subject was 2.1-fold above baseline. The expression levels of NK cell Ki67 in all four subjects were close to 100% after administration. Post-dose peripheral expansion of NK cells was an average of 3.9-fold above baseline on Day 3 in all four subjects; one subject was 5.0-fold above baseline on Day 3. From cycle 1 to cycle 2, there was no change in PK parameters. No anti-drug antibodies were detected in the first three subjects; cycle 5 was measured for two subjects and cycle 4 was measured for one subject.

During cycles 1 and 2, serum IFNγ, IL-6 and IL-5 levels were measured at 1, 2 and 3 days post-dose. Means and ranges are shown in Table 2. The highest value of the range was observed for all subjects 1 day after dosing. Table 2 Summary of Safety / Toxicity Biomarkers - Cytokines Cohort 1 : Q3W , 8 µg/kg subjects IFNγ pg/mL mean ( range ) BLQ＜3.5 IL-6 pg/mL mean ( range ) BLQ＜1.3 IL-5 pg/mL mean ( range ) BLQ＜8.8 2001-0001 29.6 (BLQ-66.9) 2.6 (BLQ-6.5) BLQ (All points BLQ) 2001-0002 19.9 (3.7-90.4) 3.1 (1.33-11.6) BLQ (All points BLQ) 2001-0003 21.3 (5.6-53.5) 3.3 (BLQ-5.96) BLQ (All points BLQ) 2001-0004 38.1 (BLQ-60.8) 4 (BLQ-6.5) BLQ (All points BLQ)

The measured cytokine levels are shown graphically in Figure 36 .

In Teachey et al., Cancer Discov. 2016; 6(6); 664-79, in patients with acute lymphoblastic leukemia treated with CAR-T cells, severe cytokine release syndrome (CRS level 4 or 5) was associated with Non-severe cytokine release syndrome (CRS levels 0–3) were associated with higher values for each of the three cytokines measured in this study. The data of Teachey et al. for IFNγ, IL-6 and IL-5 (expressed as median (range)) are reproduced in Table 3. Table 3 reports IFNγ , IL-6 and IL-5 levels in relation to CRS levels 0-3 and 4-5 . CRS level IFNγ pg/mL IL-6 pg/mL IL-5 pg/mL 0-3 34.1 (2.14-8,233) 122 (0.40-20,892) 4.25 (0.39-264) 4-5 3,119 (160-15,482) 8,309 (580-102,476) 15.3 (1.71-333)

Therefore, the results in Table 2 and Figure 36 are consistent with the absence of severe CRS.

IL-2 conjugates showed encouraging PD profiles and were generally well tolerated. Taken together, these results are considered to support non-α-preferential activity of IL-2 conjugates, with a tolerable safety profile, encouraging PD and preliminary evidence of activity in patients with immunosensitive tumors. Cohort 2 : 16 μg/kg [Q3W] dosed.

This example reports the results of up to 6 individuals with malignant solid tumors who received an IL-2 conjugate of Q3W at a dose of 16 μg/kg for at least 2 cycles. After the first administration, one subject's CD8+ T effector cells expanded peripherally by 4.1-fold after administration; the average expansion of the three patients was 2.2-fold. Post-dose peripheral expansion of NK cells was 4-fold above baseline in all three subjects; one subject was 11.4-fold above baseline, with a mean of 7.2-fold.

During cycles 1 and 2, serum IFNγ, IL-6 and IL-5 levels were measured at 1, 2 and 3 days post-dose. Means and ranges are shown in Table 4. The highest value of the range was observed 1 day after dosing for the indicated 3 subjects. Table 4 Safety / Toxicity Biomarkers - Cytokines Summary Cohort 2 : Q3W , 16 µg/kg subjects IFNγ pg/mL mean ( range ) BLQ＜3.5 IL-6 pg/mL mean ( range ) BLQ＜1.3 IL-5 pg/mL mean ( range ) BLQ＜8.8 1002-0005 91 (BLQ-349) 29.2 (3-114) BLQ (All points BLQ) 1004-0006 196.8 (BLQ-817) 21 (1.5-112) BLQ (All points BLQ) 1002-0007 190.1 (5.4-641) 62.5 (BLQ-153) BLQ (All points BLQ)

The measured cytokine levels of the 4 subjects are shown in Figure 37 . These results are also consistent with the absence of severe CRS.

Eosinophil counts of cohorts 1-2 were measured by FACS and CBC ( FIG. 38A - FIG. 38D ). In patients with IL-2-induced eosinophilia, measurements were consistently below 2328- Range of 15958 eosinophils/μL. Peripheral lymphocyte counts were also measured for cohorts 1 and 2 ( FIG. 39A- FIG. 39D ).

Efficacy biomarkers for cohorts 1 and 2 . Peripheral CD8+ T _eff counts were measured for cohorts 1 and 2 ( FIG. 40A- FIG. 40D ). Prolonged CD8+ expansion over baseline (eg, greater than or equal to a 2-fold change) was observed in some subjects 3 weeks after prior dosing. The percentage of Ki67 expressing CD8+ T _eff cells in cohorts 1 and 2 was also measured ( FIG. 41A- FIG. 41B ).

Peripheral memory CD8+ counts are shown in Figures 42A - 42B . Peripheral NK cell counts are shown in Figures 43A-D. Prolonged NK cell expansion over baseline (eg, greater than or equal to a 5-fold change) was observed in some subjects 3 weeks after prior dosing. The percentage of Ki67 expressing NK cells in cohorts 1 and 2 was also measured ( FIG. 44A- FIG. 44B ).

Figures 45A - 45B show peripheral CD4+ T _reg counts for cohorts 1 and 2. The percentage of Ki67-expressing CD4+ T _reg cells for cohorts 1 and 2 was also measured ( FIG. 46A -FIG. 46B ).

Summary of Results; Discussion. Subjects discussed above who received the 8 μg/kg dose had post-dose CD8+ Ki67 expression levels above 60% (65%-80%) and peripheral expansion of CD8+ T effector (Teff) cells averaged 1.53-fold higher than baseline. The Ki67 expression level of NK cells was also close to 100% in all 4 subjects after administration, and on day 3, the peripheral expansion of NK cells was on average 3.9 times higher than the baseline. Of the 3 subjects discussed above who received the 16 μg/kg dose, 1 post-dose peripheral expansion of CD8+ Teff cells was 4.1-fold greater than baseline at day 7; mean expansion for 3 subjects 2.2 times. No anti-drug antibodies (IL-2 or PEG) were observed, and IL-5 and IL-6 levels did not rise meaningfully. In addition, the PK data did not indicate a decrease in AUC from cycle 1 to cycle 2 (data not shown).

No dose-limiting toxicities were reported at either dose, and no treatment-related adverse events (TRAEs) or treatment-related serious AEs leading to discontinuation were reported.

The TEAEs for the 10 subjects who received the Q3W 8 or 16 μg/kg dose are detailed in Table 5. There were no grade 5 TEAEs. Grade 4 events occurred in two subjects (one with an increase in AST and one with a decrease in lymphocyte count). Table 5 Treatment Emerging Adverse Events ( TEAEs ) IL-2 conjugate 8 µg/kg Q3W (N = 4) IL-2 conjugate 16 µg/kg Q3W (N = 6) Major system organ class preference, n (%) all grades Grading≥3 _ all grades Grading≥3 _ Number of participants with TEAE 4 (100) 1 (25.0) 6 (100) 2 (33.3) Infection and Infestation 0 0 0 0 Blood and Lymphatic System Disorders 1 (25.0) 0 2 (33.3) 0 immune system disorder 0 0 0 0 Metabolic and Nutritional Disorders 0 0 1 (16.7) 0 mental disorder 0 0 0 0 nervous system disorder 0 0 2 (33.3) 0 heart disorder 1 (25.0) 0 2 (33.3) 0 Vascular disorders 1 (25.0) 0 1 (16.7) 0 Respiratory, thoracic and mediastinal disorders 2 (50.0) 0 1 (16.7) 0 Gastrointestinal disorders 4 (100) 0 4 (66.7) 0 Skin and Subcutaneous Tissue Disorders 4 (100) 1 (25.0) 2 (33.3) 0 Musculoskeletal and connective tissue disorders 1 (25.0) 0 0 0 Kidney and urinary system disorders 0 0 0 0 Systemic disorders and administration site conditions 4 (100) 0 5 (83.3) 0 an examination 0 0 5 (83.3) 2 (33.3) Injury, poisoning and surgical complications 0 0 0 0

TEAE mainly consists of flu-like symptoms, nausea or vomiting. TEAEs resolved with accepted standard of care. Treatment-related AEs were transient. AEs of fever, hypotension, and hypoxia were not associated with elevated IL-5/IL-6 cytokines. No significant effect on vital signs, no QTc prolongation or other cardiotoxicity. Thus, the IL-2 conjugate exhibited encouraging PD profiles and was generally well tolerated. The in vivo half-life of the IL-2 conjugate was determined to be about 10 hours. Taken together, these results are considered to support non-α-preferential activity of IL-2 conjugates, with a tolerable safety profile, encouraging PD and preliminary evidence of activity in patients with immunosensitive tumors.

The result of the selected individual. One subject with prostate adenocarcinoma received 10 cycles of Q3W at 16 μg/kg and showed stable disease (24% reduction after two cycles). The subject withdrew from treatment after cycle 10 due to elevated PSA.

One subject with non-small cell lung cancer received at least 6 cycles of Q3W 16 μg/kg and showed stable disease (17.9% reduction after 5 cycles).

Anti-drug antibodies ( ADA ). Anti-drug antibodies (ADA) were assayed in samples from treated subjects after each dosing period. Anti-PEG autoantibodies were detected by direct immunoassay (limit of detection: 36 ng/mL). A bridged MesoScale Discovery ELISA with a labeled form of the IL-2 conjugate had a detection limit of 4.66 ng/mL. Additionally, a cell-based assay for neutralizing antibodies against the IL-2 conjugate was performed using the CTLL-2 cell line with STAT5 phosphorylation as readout (limit of detection: 6.3 μg/mL).

Samples were collected and analyzed after each dose cycle from two subjects who received 5 dose cycles and one subject who received 4 dose cycles. Assay-specific cut-off points were determined during assay quantification with a signal to negative ratio of 1.09 or higher for the IL-2 conjugate ADA assay and 2.08 for the PEG ADA assay. Confirmatory testing was performed on samples that gave positive or indeterminate results in the IL-2 conjugate assay in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in blocking solution) Assay samples and controls. A confirmatory test was performed on samples giving positive or indeterminate results in the PEG assay, where samples were assayed in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in 6% horse serum) and contrast. Samples were considered "confirmed" if, during the detection step, the absorbance signal of the sample was suppressed by equal to or greater than the assay-specific cut-off point determined during assay quantification (14.5% for IL-2 conjugates, or 42.4% for PEG) . No confirmed ADA was detected against IL-2 conjugates or PEG (data not shown). Example 3. Clinical study of biomarker effects following administration of IL-2 conjugates and anti- PD-1 antibodies.

A study was performed to characterize the immunological impact of in vivo administration of the IL-2 conjugates described herein in combination with an anti-PD-1 antibody (in this study pembrolizumab). The same IL-2 conjugate as used in Example 2 was used in this study. IL-2 conjugates and pembrolizumab were administered every 3 weeks [Q3W] for 30 minutes by IV infusion. Analysis of the effect on the following biomarkers as surrogate predictors of safety and/or efficacy: Eosinophilia (elevated peripheral eosinophil count): IL-2 associated with vascular leak syndrome (VLS) Cell replacement marker of induced cell (eosinophil) proliferation; Interleukin 5 ( IL-5 ) : IL-2-induced priming of type 2 innate lymphocytes and leading to hypereosinophilia and potential VLS Interleukin 6 ( IL-6 ) : A cytokine replacement marker for IL-2-induced cytokine release syndrome (CRS); and Interferon gamma ( IFN-γ ) : IL-2-induced cytokine replacement marker of CD8+ cytotoxic T lymphocytes and NK cell priming.

The effect of cell count on the following biomarkers was analyzed as a surrogate predictor of anti-tumor immune activity: Peripheral CD8+ effector cells: a marker of IL-2-induced proliferation of these target cells in the periphery, which after infiltration becomes an induction of possible therapeutic potential Surrogate markers of response; peripheral CD8+ memory cells: a marker of IL-2-induced proliferation of these target cells in the periphery, after infiltration a surrogate marker for the induction of potentially therapeutic and maintenance memory populations that may persist; peripheral NK cells: A marker of IL-2-induced proliferation of these target cells in the periphery, after infiltration a surrogate marker for induction of a potentially rapid therapeutic response; and Peripheral CD4+ regulatory cells: a marker of IL-2-induced proliferation of these target cells in the periphery After infiltration, they become surrogate markers for inducing immunosuppressive TME and counteracting effector-based therapeutics.

Subjects were male or female aged ≥ 18 years at screening. All subjects have been previously treated with anticancer therapy and meet at least one of the following: treatment-related toxicity resolved to grade 0 or 1 according to NCI CTCAE v5.0 (except alopecia); or treatment-related toxicity according to NCI CTCAE v5.0 Regression to at least grade 2, prior approval by medical monitor. The most common tumors include cervical cancer, squamous cell carcinoma of the head and neck, basal cell carcinoma, melanoma, and non-small cell lung cancer.

Subjects also met the following criteria: Provide informed consent. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Investigator-determined life expectancy greater than or equal to 12 weeks. Histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors. Advanced or metastatic solid tumors who refuse standard of care; or where no reasonable standard of care would confer clinical benefit; or who are intolerable, ineffective, or unavailable to standard therapy. Disease measurable according to RECIST v1.1. Appropriate laboratory parameters include: absolute lymphocyte count ≥ 0.5 times the lower limit of normal; platelet count ≥ 100 × 10 ⁹ /L; hemoglobin ≥ 9.0 g/dL (without growth factors or blood transfusion within 2 weeks; ESA and CSF administered 1 week clearance is sufficient); absolute neutrophil count ≥ 1.5 × 10 ⁹ /L (growth factor-free for 2 weeks); prothrombin time (PT) and partial thromboplastin time (PTT) ≤ 1.5 times upper limit of normal (ULN); aspartate transaminase (AST) and alanine transaminase (ALT) ≤ 2.5 times ULN, unless liver metastases may be ≤ 5 times ULN; total bilirubin ≤ 1.5 × ULN. Premenopausal women and women less than 12 months postmenopausal had a negative serum pregnancy test within 7 days before starting study treatment.   Cohorts treated with 8 μg/kg and 16 μg/kg doses

Q3W dosing. Fourteen adults (9 [64.3%] male, 5 [35.7%] female, 9 [64.2%] Caucasian) with advanced or metastatic solid tumors and aged 29-74 years received a) IL-2 conjugate at 8 μg/kg dose IV Q3W or 16 μg/kg dose IV Q3W and b) pembrolizumab at 200 mg dose IV Q3W for at least one cycle. Here and throughout Example 3, drug mass per kg of subject (e.g., 8 μg/kg) refers to IL-2 mass excluding PEG and linker mass. The results below are for subjects who received 8 μg/kg IV Q3W and pembrolizumab (4 subjects) or 16 μg/kg IV Q3W and pembrolizumab (6 subjects subjects), who received 2-19 cycles of treatment.

Two subjects who received 8 μg/kg IL-2 conjugate and pembrolizumab had confirmed partial responses (PR; 1 PD-1 naïve basal cell carcinoma, 1 head and neck squamous cell carcinoma, received Previous anti-PD-1), lasted for 22+ months. One subject (non-small cell lung cancer) who received 16 μg/kg IL-2 conjugate and pembrolizumab had stable disease for approximately 6 months. Six subjects had progressive disease (at the 6-week assessment); one subject had initially stable disease (at the 6-week assessment; subsequently progressive disease). Four subjects receiving 8 μg/kg IL-2 conjugate and pembrolizumab had post-dose increases in CD8+ Ki67 expression levels (15%-70%).

A 59-year-old man with head and neck squamous cell carcinoma received 8 μg/kg IL-2 conjugate and pembrolizumab for 30+ cycles and had a confirmed partial response (39% drop after 8 cycles ; decreased by 47% after 11 periods). The subject had previously received 4 lines of systemic therapy including 2 anti-PD1 treatments; the best response to anti-PD1 treatment was stable disease.

A 50-year-old man with basal cell carcinoma received 8 μg/kg IL-2 conjugate and pembrolizumab for 30 cycles with a confirmed partial response (50% drop after 2 cycles, 8 cycles down 80%). The subject had previously undergone surgery and radiation therapy.

The largest tumor responses among other patients with immunosensitive tumors were found to be melanoma (23% and 11% growth), basal cell carcinoma (4% growth) and non-small cell lung cancer (29% reduction).

In subjects receiving 8 μg/kg IL-2 conjugate and pembrolizumab, the peak peripheral expansion of CD8+ T effector cells was on average 2.06-fold above baseline. The expression levels of NK cell Ki67 in all four subjects were close to 100% after administration. On day 3, the post-administration peak peripheral expansion of the subject's NK cells was on average 6.73 times higher than the baseline. In subjects receiving 16 μg/kg IL-2 conjugate and pembrolizumab, peak peripheral expansion of CD8+ T effector cells was on average 3.71-fold higher than baseline.

Efficacy biomarkers. Data related to efficacy biomarkers are based on available data from 10 subjects (4 subjects received 8 μg/kg IL-2 conjugate; 6 subjects received 16 μg/kg IL-2 conjugate things). Peripheral CD8+ T _eff cell counts were measured ( FIG. 10A -FIG. 10C ). Prolonged CD8+ expansion over baseline (eg, greater than or equal to a 1.5-fold change) was observed in some subjects 3 weeks after prior dosing. The percentage of CD8+ T _eff cells expressing Ki67 was also measured ( FIG. 11 ).

Peripheral NK cell counts are shown in Figures 12A - 12C . Prolonged NK cell expansion over baseline (eg, greater than or equal to a 2-fold change) was observed in some subjects 3 weeks after prior dosing. The percentage of NK cells expressing Ki67 was also measured ( Figure 13 ).

Figures 14A - 14C show peripheral CD4+ T _reg counts. The percentage of CD4+ T _reg cells expressing Ki67 was also measured ( FIG. 15 ).

Eosinophil counts were measured ( Figure 16A -Figure 16C ). In patients with IL-2-induced eosinophilia, measurements were consistently below 2328- Range of 15958 eosinophils/μL. Levels of IFN-γ, IL-5 and IL-6 were also measured ( Figure 17A - Figure 17D ). Measurements showed induction of IFN-γ but less IL-5 and IL-6 (cytokines associated with VLS and CRS, respectively).

Figure 18A and Figure 18B show the average concentration of IL-2 conjugate administered at a dose of 8 μg/kg after 1 and 2 cycles, respectively. Figure 18C and Figure 18D show the average concentration of IL-2 conjugate administered at a dose of 16 μg/kg after 1 and 2 cycles, respectively.

Anti-drug antibodies ( ADA ). Anti-drug antibodies (ADA) were assayed in samples from treated subjects after each dosing period. Anti-PEG autoantibodies were detected by direct immunoassay (limit of detection: 36 ng/mL). A bridged MesoScale Discovery ELISA with a labeled form of the IL-2 conjugate had a detection limit of 4.66 ng/mL. Additionally, a cell-based assay for neutralizing antibodies against the IL-2 conjugate was performed using the CTLL-2 cell line with STAT5 phosphorylation as readout (limit of detection: 6.3 μg/mL).

Samples from four subjects were collected and analyzed after each dosing cycle, with 2 patients receiving 2 cycles and the other two patients receiving 10 or 11 cycles. Assay-specific cut-off points were determined during assay quantification with a signal to negative ratio of 1.09 or higher for the IL-2 conjugate ADA assay and 2.08 for the PEG ADA assay. Confirmatory testing was performed on samples that gave positive or indeterminate results in the IL-2 conjugate assay in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in blocking solution) Assay samples and controls. A confirmatory test was performed on samples giving positive or indeterminate results in the PEG assay, where samples were assayed in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in 6% horse serum) and contrast. Samples were considered "confirmed" if, during the detection step, the absorbance signal of the sample was suppressed by equal to or greater than the assay-specific cut-off point determined during assay quantification (14.5% for IL-2 conjugates, or 42.4% for PEG) . No confirmed ADA was detected against IL-2 conjugates or PEG (data not shown).

Summary of Results; Discussion. The CD8+ Ki67 expression levels of all subjects increased after administration ( Figure 11 ), and the peripheral expansion of CD8+ T effector (Teff) cells was 1.95 times higher than the baseline on average. Ki67 expression levels of NK cells also increased in all 4 subjects after administration ( Figure 13 ), and on day 3, the peripheral expansion of NK cells was on average 6.73 times higher than baseline. IL-5 and IL-6 levels did not rise meaningfully.

An AE is any adverse medical event, regardless of cause, in a clinical study subject administered a drug product. Dose-limiting toxicities were defined as AEs occurring within ± 1 day of treatment cycle Day 1 to Day 29 (inclusive) that were not clearly or indisputably related to extrinsic causes only, and that met at least one of the following criteria: 3 Grade 4 neutropenia (absolute neutrophil count < 1000/mm ³ > 500/mm ³ ) lasting ≥ 7 days, or Grade 4 neutropenia of any duration • Grade 3+ Febrile neutropenia • Grade 4 + thrombocytopenia (platelet count < 25,000/mm ³ ) • Grade 3 + thrombocytopenia (platelet count < 50,000-25,000/mm ³ ) for ≥ 5 days, or Associated with clinically significant bleeding or requiring platelet transfusion • Failure to meet recovery criteria within 10 days of an absolute neutrophil count of at least 1,000 cells/ ^mm3 and a platelet count of at least 75,000 cells/ ^mm3 • Any other classification 4+ hematological toxicity lasting ≥ 5 days • Grade 3+ ALT or AST and bilirubin >2 times ULN, no evidence of cholestasis or other causes such as viral infection or other drugs (i.e. Hy's law) • Premedication occurred Grade 3 Infusion-Related Reactions; Grade 4 Infusion-Related Reactions Grade 3 Vascular Leak Syndrome, defined as hypotension associated with fluid retention and pulmonary edema Grade 3+ Anaphylaxis Grade 3+ Hypotension Grade 3+AEs Does not resolve to <Grade 2 within 7 days of starting standard of care medical management • Grade 3 + cytokine release syndrome The following exceptions apply for non-hematologic AEs: • Grade 3 fatigue, nausea, vomiting, or diarrhea within ≤ 3 days Resolution by best medical management to ≤ Grade 2 • Grade 3 fever (defined as >40ºC for ≤ 24 hours) • Grade 3 infusion-related reaction, occurred in the absence of premedication; subsequent doses should use premedication, AND if response recurs, DLT Grade 3 arthralgia or rash that resolves to ≤ Grade 2 within 7 days of starting standard-of-care medical management (eg, systemic corticosteroid therapy) if subject was Grade 1 or For grade 2 ALT or AST elevation (considered to be indirectly caused by liver metastasis), the grade 3 elevation must also be ≥ 3 times baseline and last for > 7 days.

A serious AE was defined as any AE leading to any of the following outcomes: death; life-threatening AE; hospitalization or prolongation of current hospitalization; persistent or severe loss or severe impairment of ability to perform normal life functions; Anomalies/Birth Defects. A medically important event that is likely not to result in death, is life-threatening, or requires hospitalization may be considered serious when, based on appropriate medical judgment, it may endanger the subject and may require medical or surgical intervention to prevent one of the outcomes listed above event. Examples of such medical events include allergic bronchospasm requiring intensive treatment in the emergency department or at home, blood dyscrasias or convulsions that do not result in hospitalization, or development of drug dependence or substance abuse.

No dose-limiting toxicities were reported at either dose, and no treatment-related adverse events (TRAEs) leading to discontinuation occurred. A TRAE resulted in a dose reduction. Five treatment-related serious AEs were reported in three of the patients treated with the 16 μg/kg dose of IV Q3W.

According to SOC, the most common TRAEs (>2 patients) of all grades included general disturbance and administration status, work-up, metabolism and nutrition, nervous system disturbance, respiratory, thoracic and mediastinal disturbance, vascular disturbance, skin and subcutaneous disturbance, blood and Lymphatic system disorders, cardiac disorders, gastrointestinal disorders, immune system disorders, infections and infestations, and musculoskeletal disorders. TEAEs expressed in preferred terms are detailed in Table 6. Table 6 8 µg/kg IL-2 conjugate Q3W+pembrolizumab ( N=4) 16 µg/kg IL-2 conjugate Q3W+pembrolizumab ( N=10) Major system organ class preference, n (%) all grades Grading≥3 _ all grades Grading≥3 _ Number of participants with TEAE 3 (75.0) 0 10 (100) 6 (60.0) Infection and Infestation 1 (25.0) 0 1 (10.0) 0 Blood and Lymphatic System Disorders 0 0 2 (20.0) 1 (10.0) immune system disorder 0 0 2 (20.0) 1 (10.0) Endocrine disorders 0 0 2 (20.0) 0 Metabolic and Nutritional Disorders 1 (25.0) 0 4 (40.0) 1 (10.0) mental disorder 1 (25.0) 0 0 0 nervous system disorder 2 (50.0) 0 3 (30.0) 0 Eye disorders 1 (25.0) 0 0 0 heart disorder 0 0 2 (20.0) 0 Vascular disorders 1 (25.0) 0 3 (30.0) 1 (10.0) Respiratory, thoracic and mediastinal disorders 1 (25.0) 0 3 (30.0) 1 (10.0) Gastrointestinal disorders 1 (25.0) 0 4 (40.0) 0 Hepatobiliary disorders 0 0 1 (10.0) 0 Skin and Subcutaneous Tissue Disorders 1 (25.0) 0 3 (30.0) 0 Musculoskeletal and connective tissue disorders 1 (25.0) 0 3 (30.0) 0 Systemic disorders and administration site conditions 3 (75.0) 0 10 (100) 0 an examination 2 (50.0) 0 8 (80.0) 4 (40.0) Injury, poisoning and surgical complications 1 (25.0) 0 1 (10.0) 0

Treatment-related AEs were transient and resolved with accepted standard of care. AEs of fever, hypotension, and hypoxia were not associated with elevated IL-5/IL-6 cytokines. Cumulative toxicity, end-organ toxicity, vascular leak syndrome, or eosinophilia were not observed. IL-5 levels were maintained at or below the minimum detection level. One subject suffered from G2 hypotension, which resolved with hydration. One subject had G3 hormone release syndrome (fever + hypotension requiring vasopressors; subject had baseline orthostatic hypotension). One subject experienced recurrent G2 hormone-releasing syndrome with fever and hypoxia (the patient had underlying COPD that resolved with supportive care management including 1 dose of tociluzimab). The subject's dose was reduced to 8 μg/kg; the subject then developed G2 pneumonia and was rechallenged after improving to G1. Subsequently, the subject developed recurrent G3 pneumonia without receiving further therapy. No significant effect on vital signs, no QTc prolongation or other cardiotoxicity. Thus, IL-2 conjugates in combination with pembrolizumab showed encouraging PD profiles and were generally well tolerated with no discontinuations due to TRAEs. The in vivo half-life of the IL-2 conjugate was determined to be about 10 hours. Taken together, these results are considered to support non-α-preferential activity of IL-2 conjugates, a tolerable safety profile in combination with pembrolizumab, and encouraging preliminary evidence of PD and activity in patients with immunosensitive tumors .   Cohort treated with 24 μg/kg dose

Ten individuals (male [100%], 6 [60.0%] Caucasian) with a median age of 61 years (range 46-68) with advanced or metastatic solid tumors received Q3W at a dose of 24 μg/kg IL-2 conjugates. Tumor types include lung cancer, basal cell carcinoma, and colon cancer.

Each subject was sequentially treated with: a) IL-2 conjugate administered by IV infusion at a dose of 24 μg/kg for 30 minutes, and b) pembrolizumab administered IV at a dose of 200 mg anti. Treatment was administered every 3 weeks [Q3W]. The effect of IL-2 conjugates at doses of 8 μg/kg and 16 μg/kg on the same biomarkers described above was analyzed as a surrogate predictor of safety and/or efficacy. Subjects in these studies met the same criteria as subjects treated with the 8 μg/kg and 16 μg/kg doses.

All 10 subjects experienced at least one TEAE, and 7 of 10 subjects (70.0%) experienced at least 1 grade 3-4 related TEAE (1 grade 3 and 6 grade 4). One Grade 3 ALT/AST elevation (also with Grade 3 hypophosphatemia), G2 hyperbilirubinemia in the case of G2 CRS, and five Grade 4 lymphocyte count decreases (once subject had 3 Grade AST/ALT elevation, grade 2 hyperbilirubinemia-DLT and grade 2 CRS) and 1 G4 lymphopenia. Lymphocyte count recovered to at least grade 3 within 48 hours.

Six subjects experienced related SAEs: one grade 1 fever in a subject with adrenal insufficiency requiring steroid adjustment, and one grade 2 related SAE associated with grade 3 AST/ALT elevation and G2 hyperbilirubinemia Grade cytokine release syndrome (fever and hypotension requiring fluids and dexamethasone). In addition, one subject developed G2 hypotension and received supportive care, and one subject experienced an infusion-related reaction during C1D1 followed by G2 CRS after Cycle 2 with fever, chills, and hypotension and received support Sexual care, another subject who developed a G3 infusion-related reaction during C1, followed by cytokine release syndrome G1 during Cycle 2, and one subject who developed G2 CRS during Cycle 2 received supportive care. There was one DLT: Subject had Grade 3 AST/ALT elevation and Grade 2 hyperbilirubinemia associated with Grade 2 CRS (fever and hypotension requiring hydration and dexamethasone). For this subject, the dose of C2D1 was reduced. There were discontinuations due to TEAEs. TEAEs are detailed in Table 7. Table 7 Treatment-emergent adverse events (TEAE) ( n=10 ) 24 µg/kg Q3W+pembrolizumab ( N=10) Major system organ class preference, n (%) all grades Grading≥3 _ Number of participants with TEAE 10 (100) 7 (70.0) Infection and Infestation 1 (10.0) 0 Blood and Lymphatic System Disorders 3 (30.0) 1 (10.0) immune system disorder 3 (30.0) 0 Endocrine disorders 0 0 Metabolic and Nutritional Disorders 3 (30.0) 1 (10.0) mental disorder 1 (10.0) 0 nervous system disorder 1 (10.0) 0 Eye disorders 0 0 heart disorder 0 0 Vascular disorders 2 (20.0) 0 Respiratory, thoracic and mediastinal disorders 0 0 Gastrointestinal disorders 5 (50.0) 0 Hepatobiliary disorders 1 (10.0) 0 Skin and Subcutaneous Tissue Disorders 3 (30.0) 0 Musculoskeletal and connective tissue disorders 2 (20.0) 0 Systemic disorders and administration site conditions 8 (80.0) 1 (10.0) an examination 6 (60.0) 6 (60.0) Injury, poisoning and surgical complications 4 (40.0) 1 (10.0)

The following related events were reported: one time in the setting of grade 2 CRS (fever, hypotension [BP97/56 mm Hg] and hypoxemia [SpO2 92%]), grade 3 AST/ALT and grade 2 bilirubin ( DLT), received a fluid bolus, supplemental oxygen, and dexamethasone, requiring a dose reduction of C2D1 to resolve; one patient had fever, chills, chills, and hypoxemia (92%), requiring supportive care and oxygen (C2D1 ); one grade 3 AST/ALT (C2D8) in the setting of alcoholism, presumably related to IL-2 conjugate and pembrolizumab, without other symptoms; and three grade 4 lymphocyte count drops.

Efficacy biomarkers. Data related to efficacy biomarkers are based on available data from 6 subjects who received 24 μg/kg IL-2 conjugate. Peripheral CD8+ T _eff cell counts were measured ( FIG. 19 ), and peripheral NK cell counts are shown in FIG. 20 . Figure 21 shows peripheral CD4+ T _reg cell counts, and Figure 22 shows peripheral eosinophil counts.

Figure 23A and Figure 23B show the average concentration of IL-2 conjugates after 1 and 2 cycles, respectively.

Figure 24 shows cytokine levels (IFN-γ, IL-6 and IL-5).

Thus, IL-2 conjugates in combination with pembrolizumab showed encouraging PD profiles and were generally well tolerated with no discontinuations due to TRAEs. Taken together, these results are considered to support non-α-preferential activity of IL-2 conjugates, a tolerable safety profile in combination with pembrolizumab, and encouraging preliminary evidence of PD and activity in patients with immunosensitive tumors . Example 4. Administration of different doses of IL-2 conjugates to subjects with skin cancer.

A total of 6 individuals with skin cancer received the IL-2 conjugate used in the study described in Example 2. Three individuals with melanoma received an IL-2 conjugate at a dose of 8 μg/kg Q3W, one individual with melanoma received an IL-2 conjugate at a dose of 24 μg/kg Q3W, and one with basal cells One individual with basal cell carcinoma (BCC) received an 8 μg/kg dose (with pembrolizumab) of IL-2 conjugate Q3W, and one individual with basal cell carcinoma (BCC) received a dose of 16 μg/kg (with pembrolizumab) mAb together) IL-2 conjugate of Q3W, and 1 individual with basal cell carcinoma (BCC) received IL-2 conjugate at a dose of 24 μg/kg (with pembrolizumab), up to 13 each cycles (1 dose per cycle). Here and throughout Example 4, drug mass per kg subject (eg, 24 μg/kg) refers to IL-2 mass excluding PEG and linker mass.

Clinical outcomes in subjects with basal cell carcinoma were partial responses in individuals receiving the 8 μg/kg and 24 μg/kg doses and stable disease in individuals receiving the 16 μg/kg dose. Subjects receiving the 8 μg/kg dose showed a 50% reduction after two treatment cycles, which became a 66.7% reduction after five treatment cycles, and an 80% reduction after eight treatment cycles. Individuals receiving the 24 μg/kg dose had a confirmed partial response after 8 cycles, with an 85% reduction in target lesion size. The reported reduction is the sum of the product of the largest diameters of the target lesions. efficacy biomarkers

CD8+ _Teff cells . Peripheral CD8+ T _eff cell counts were determined for each of the 6 individuals. Table 8 summarizes the fold change in CD8+ T _eff cell counts normalized to pre-treatment cell counts. The data indicate that administration of IL-2 conjugates promotes CD8+ expansion over baseline (eg, up to about a 2-fold change) in subjects with melanoma or BCC.

NK cells. Peripheral NK cell counts were determined for each of the 6 individuals. Table 9 summarizes the fold change in peripheral NK cell counts normalized to pre-treatment cell counts. An increase in NK cell counts was observed in each subject.

CD4+ _Treg cells. Peripheral CD4+ T _reg cell counts were determined for each of the 6 individuals. Table 10 summarizes the fold change in peripheral CD4+ T _reg cell counts normalized to pre-treatment cell counts. The data indicate that administration of IL-2 conjugates does not significantly promote CD4+ expansion beyond baseline.

lymphocytes. Lymphocyte counts were determined for each of the 6 individuals. Table 11 summarizes the fold change in lymphocyte counts normalized to pre-treatment cell counts. The data show that administration of IL-2 conjugates promotes lymphocyte expansion over baseline (eg, up to about a 2-fold change).

Eosinophils. Eosinophil counts were determined for each of the 6 individuals. Table 12 summarizes the fold change in eosinophil counts normalized to pre-treatment cell counts. The measured values did not increase more than 5-fold.

IFN-γ , IL-5 and IL-6 . IFN-γ, IL-5 and IL-6 levels were determined for each of the 6 individuals. Cytokine levels in patients with melanoma are shown in Figure 25A . Measurements indicated induction of IFN-γ but less IL-5 and IL-6 (cytokines associated with VLS and CRS, respectively), except for one subject whose IL-6 levels increased 4 hours after treatment to about 200 pg/mL, but declined thereafter. Cytokine levels in patients with BCC are shown in Figure 25B . For these patients, measurements showed induction of IFN-γ, but less IL-5 and IL-6. Table 8. Normalized CD8+ T _eff cell counts. Treatment cycle 1 Treatment cycle 2 cycle 3 cycle 4 cycle 5 time after treatment ^a time after treatment ^a time after treatment ^a Patient (cancer type, dose μg/kg) 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 0hr 0hr 2001-0001 (melanoma, 8) 1.00 0.78 0.41 0.86 0.62 0.22 0.62 0.53 0.90 0.74 0.79 2001-0002 (melanoma, 8) 1.00 0.28 0.41 0.53 1.01 0.27 0.86 1.10 1.01 0.61 0.56 2001-0003 (melanoma, 8) 1.00 0.73 1.30 0.83 1.70 1.20 2.38 1.47 0.80 2.18 1.76 2001-0011 (melanoma, 24) 1.00 0.01 0.01 0.06 2.17 2.46 0.02 0.02 0.16 1.25 1.88 2.63 1.08 2003-0019 (BCC, 8) 1.00 0.16 0.05 0.39 1.39 2.91 1.64 0.16 0.13 0.56 1.32 1.35 1.30 1.88 2003-0035 (BCC, 16) 1.00 0.00 0.03 0.01 0.08 0.53 0.20 0.02 0.01 ^a Ohr refers to cell count determinations just prior to cycle treatment. Table 8 (continued) cycle 6 cycle 7 cycle 8 cycle 9 cycle 10 cycle 11 cycle 12 cycle 13 time after treatment ^a Patient (cancer type, dose μg/kg) 0hr 0hr 0hr 0hr 0hr 0hr 0hr 0hr 2001-0001 (melanoma, 8) 2001-0002 (melanoma, 8) 2001-0003 (melanoma, 8) 2001-0011 (melanoma, 24) 2003-0019 (BCC, 8) 1.56 1.22 2.14 2.19 1.92 2.14 2.27 2.14 2003-0035 (BCC, 16) Table 9. Normalized peripheral NK cell counts. Treatment cycle 1 Treatment cycle 2 cycle 3 cycle 4 cycle 5 time after treatment ^a time after treatment ^a time after treatment ^a Patient (cancer type, dose μg/kg) 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 0hr 0hr 2001-0001 (melanoma, 8) 1.00 0.02 0.14 4.25 0.80 0.03 0.96 3.80 1.22 1.18 1.75 2001-0002 (melanoma, 8) 1.00 0.02 0.32 2.00 1.65 0.03 0.90 3.13 2.17 1.25 2.31 2001-0003 (melanoma, 8) 1.00 0.04 0.40 4.19 2.55 0.04 0.70 3.80 4.97 3.90 6.24 2001-0011 (melanoma, 24) 1.00 0.01 0.01 0.07 4.23 2.98 0.02 0.03 0.20 2.41 2.28 3.02 0.93 2003-0019 (BCC, 8) 1.00 0.02 0.01 0.35 4.72 8.81 2.72 0.09 0.12 0.95 6.87 2.99 3.33 4.91 2003-0035 (BCC, 16) 1.00 0.01 0.00 0.03 0.64 2.30 0.57 0.01 0.01 ^a Ohr refers to cell count determinations just prior to cycle treatment. Table 9 (continued) cycle 6 cycle 7 cycle 8 cycle 9 cycle 10 cycle 11 cycle 12 cycle 13 time after treatment ^a Patient (cancer type, dose μg/kg) 2001-0001 (melanoma, 8) 2001-0002 (melanoma, 8) 2001-0003 (melanoma, 8) 2001-0011 (melanoma, 24) 2003-0019 (BCC, 8) 3.92 2.79 4.88 3.66 4.02 5.08 3.64 3.02 2003-0035 (BCC, 16) Table 10. Normalized peripheral CD4+ T _reg cell counts. Treatment cycle 1 Treatment cycle 2 cycle 3 cycle 4 cycle 5 time after treatment ^a time after treatment ^a time after treatment ^a Patient (cancer type, dose μg/kg) 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 0hr 0hr 2001-0001 (melanoma, 8) 1.00 0.41 0.44 1.25 0.79 0.14 0.94 1.15 0.80 0.73 0.67 2001-0002 (melanoma, 8) 1.00 0.27 0.45 0.79 0.82 0.30 0.71 1.18 0.71 0.52 0.63 2001-0003 (melanoma, 8) 1.00 0.35 0.86 1.45 1.60 0.34 1.44 1.54 1.26 1.24 1.74 2001-0011 (melanoma, 24) 1.00 0.06 0.03 0.09 1.80 1.96 0.06 0.02 0.30 1.17 1.27 1.68 0.65 2003-0019 (BCC, 8) 1.00 0.23 0.13 0.62 1.22 2.04 1.20 0.16 0.23 0.60 1.05 1.03 0.91 1.15 2003-0035 (BCC, 16) 1.00 0.05 0.11 0.08 0.29 0.97 0.37 0.04 0.03 ^a Ohr refers to cell count determinations just prior to cycle treatment. Table 10 (continued) cycle 6 cycle 7 cycle 8 cycle 9 cycle 10 cycle 11 cycle 12 cycle 13 time after treatment ^a Patient (cancer type, dose μg/kg) 2001-0001 (melanoma, 8) 2001-0002 (melanoma, 8) 2001-0003 (melanoma, 8) 2001-0011 (melanoma, 24) 2003-0019 (BCC, 8) 0.98 0.74 1.21 1.20 1.03 1.08 1.18 0.91 2003-0035 (BCC, 16) Table 11. Normalized lymphocyte counts. Treatment cycle 1 Treatment cycle 2 cycle 3 cycle 4 cycle 5 time after treatment ^a time after treatment ^a time after treatment ^a Patient (cancer type, dose μg/kg) 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 0hr 0hr 2001-0001 (melanoma, 8) 1.00 0.34 0.31 1.50 0.78 0.14 0.79 1.30 0.85 0.79 0.83 2001-0002 (melanoma, 8) 1.00 0.17 0.41 1.07 1.03 0.19 0.69 1.55 1.02 0.61 0.86 2001-0003 (melanoma, 8) 1.00 0.31 0.70 1.51 1.75 0.33 1.20 1.58 1.56 1.49 2.15 2001-0011 (melanoma, 24) 1.00 0.04 0.02 0.08 2.13 2.17 0.04 0.02 0.24 1.40 1.54 2.04 0.74 2003-0019 (BCC, 8) 1.00 0.23 0.11 0.53 1.63 3.22 1.57 0.18 0.19 0.61 1.85 1.44 1.40 1.92 2003-0035 (BCC, 16) 1.00 0.04 0.09 0.05 0.26 1.08 0.36 0.03 0.02 ^a Ohr refers to cell count determinations just prior to cycle treatment. Table 11 (continued) cycle 6 cycle 7 cycle 8 cycle 9 cycle 10 cycle 11 cycle 12 cycle 13 time after treatment ^a Patient (cancer type, dose μg/kg) 2001-0001 (melanoma, 8) 2001-0002 (melanoma, 8) 2001-0003 (melanoma, 8) 2001-0011 (melanoma, 24) 2003-0019 (BCC, 8) 1.57 1.21 2.04 1.88 1.79 2.07 2.00 1.57 2003-0035 (BCC, 16) Table 12. Normalized eosinophil counts. Treatment cycle 1 Treatment cycle 2 cycle 3 cycle 4 cycle 5 time after treatment ^a time after treatment ^a time after treatment ^a Patient (cancer type, dose μg/kg) 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 4 hours 24 hours 48 hours 72 hours 168 hours 0hr 0hr 0hr 2001-0001 (melanoma, 8) 1.00 0.78 0.41 0.86 0.62 0.22 0.62 0.53 0.90 0.74 0.79 2001-0002 (melanoma, 8) 1.00 0.28 0.41 0.53 1.01 0.27 0.86 1.10 1.01 0.61 0.56 2001-0003 (melanoma, 8) 1.00 0.73 1.30 0.83 1.70 1.20 2.38 1.47 0.80 2.18 1.76 2001-0011 (melanoma, 24) 1.00 0.23 0.45 1.19 1.76 1.27 0.15 0.25 1.25 1.64 1.43 1.24 0.36 2003-0019 (BCC, 8) 1.00 0.49 1.23 1.59 1.74 1.70 1.05 0.25 1.56 1.04 1.11 0.92 0.76 1.16 2003-0035 (BCC, 16) 1.00 1.51 0.45 2.40 2.82 2.54 1.05 0.28 1.85 ^a Ohr refers to cell count determinations just prior to cycle treatment. Table 12 (continued) cycle 6 cycle 7 cycle 8 cycle 9 cycle 10 cycle 11 cycle 12 cycle 13 time after treatment ^a Patient (cancer type, dose μg/kg) 2001-0001 (melanoma, 8) 2001-0002 (melanoma, 8) 2001-0003 (melanoma, 8) 2001-0011 (melanoma, 24) 2003-0019 (BCC, 8) 4.49 2.09 1.67 1.53 3.44 4.65 2.80 1.33 2003-0035 (BCC, 16) Example 5. Use of IL-2 conjugates and checkpoint inhibitors in the treatment of CT-26 tumor -bearing Balb/c mice.

，q是3，並且W是具有30 kDa的平均分子量的甲氧基線性PEG基團，和/或包含式 (I) 的結構的化合物，其中Y是CH ₂，並且Z是

，q是3，並且W是具有30 kDa的平均分子量的甲氧基線性PEG基團。 使用其中首先製備具有SEQ ID NO: 4的蛋白質的方法製備化合物，其中位置65的脯胺酸被 N6-((2-疊氮基乙氧基)-羰基)-L-離胺酸AzK替代。 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 4) 然後允許含有AzK的蛋白質在點擊化學條件下與包含平均分子量為30 kDa的甲氧基線性PEG基團的DBCO反應，隨後使用標準程序純化和配製。 在本研究中使用包含SEQ ID NO: 3的IL-2接合物“IL-2_P65[AzK_PEG30kD]”（在本文和附圖中也稱為“化合物A”）： APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO : 3) where [AzK_PEG30kD] is N6-((2-azidoethoxy)-carbonyl)-L-lysine stably conjugated to PEG via DBCO-mediated click chemistry to form The compound of the structure of the above formula (I), wherein Z is CH ₂ , Y is

, q is 3, and W is a methoxy linear PEG group having an average molecular weight of 30 kDa, and/or a compound comprising a structure of formula (I), wherein Y is CH ₂ , and Z is

, q is 3, and W is a methoxy linear PEG group with an average molecular weight of 30 kDa. Compounds were prepared using a method in which a protein having SEQ ID NO: 4 was first prepared, wherein the proline at position 65 was replaced by N 6-((2-azidoethoxy)-carbonyl)-L-lysine AzK . APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 4) was then allowed to react under click chemistry conditions with a linear reaction PEG group containing a methoxyl CO group with an average molecular weight of 30 kDa and subsequent purification using standard procedures for DBCO.

Compound A studies as monotherapy and in combination with anti-PD-1 antibodies were performed in Balb/c female mice. Balb/c female mice weighing an average of 16 g–21 g at 6–8 weeks of age were purchased from Jackson Laboratories (Sacramento, CA) for studies 1 and 2. Average weight of 18–22 g at 7–8 weeks of age Balb/c female mice of g were purchased from Taconic Biosciences of HD Biosciences and used in Study 3. Cryopreserved vials of CT-26 colon carcinoma cells were purchased from the American Tissue Type Collection (ATCC, Manassas, VA). Cells were thawed and cultured according to the manufacturer's protocol. On the day of tumor cell inoculation, cells were washed in serum-free medium, counted, and resuspended in cold serum-free medium at a concentration of 250,000 (Studies 1 and 2) or 300,000 (Study 3) viable cells/0.1 mL. CT-26 cells (0.1 mL) were injected subcutaneously into the flank of individual mice and tumors were allowed to grow.

For Studies 1 and 2, in which a combination of compound A and an anti-PD-1 antibody was used, the antibody used was anti-mouse PD-1 (BioXcell; RMP1-14) and the control antibody was an IgG1 isotype antibody (BioXcell; catalog No. BP0089, Lot No. 2A3). For Study 3, in which an anti-PD-1 antibody was used, the antibody used was anti-mouse PD-1 (BioXcell; Cat. No. BP0146, RMP1-14, Lot No. 695318A1), and the control antibody was an IgG1 isotype antibody (BioXcell; Cat. No. BP0089, lot number 2A3).

The lyophilized compound A was reconstituted into a 10 mg/mL stock solution with 0.1 M acetic acid. Then further dilute to working concentration with 1x phosphate-buffered saline (PBS). Compounds were reconstituted and diluted within 1 hour of dosing to animals and kept on ice until dosing. Store lyophilized compounds at -80ºC until use. Store the medium at 4ºC.

Three separate efficacy studies were performed using CT-26 tumor bearing Balb/c mice. The design of Study 1, which evaluated the dose-dependent efficacy of Compound A as a single agent, is summarized in Table 13. Tables 14 and 15 summarize the design of Studies 2 and 3, respectively, which evaluated the efficacy of Compound A in combination with an anti-PD-1 antibody. The route of administration of Compound A was intravenous (IV). Mice were administered IV via the tail vein. Antibodies were administered intraperitoneally (IP). All doses were administered based on the individual body weight of each animal obtained just prior to each dose. Details of the dosing regimen are described below. Table 13. Study #1: Control and test treatment groups in CT-26 tumor bearing mice. potion Dose (mg/kg) way, program number of mice Vehicle control 0 IV, QWx3 10 Compound A 0.3 IV, QWx3 10 Compound A 0.3 IV, Q2Wx2 10 Compound A 1 IV, QWx3 10 Compound A 1 IV, Q2Wx2 10 Compound A 3 IV, QWx3 10 Compound A 3 IV, Q2Wx2 10 IV = intravenous; QWx3 = weekly for 3 doses; Q2Wx2 = every 2 weeks for 2 doses. Table 14. Study #2: Control and test treatment groups in CT-26 tumor bearing mice. potion Dose (mg/kg) way, plan number of mice Vehicle control + IgG isotype control 0 + 10 IV, QWx3 + IP, BIWx3 14 Compound A 3 IV, QWx3 14 Compound A 6 IV, QWx3 14 anti-PD-1 antibody 10 IP, BIWx3 14 Compound A+anti-PD-1 antibody 6 + 10 IV, QWx3 + IP, BIWx3 14 BIWx3=twice a week for 3 weeks, a total of 6 doses; IP=intraperitoneal; IV=intravenous; QWx3=once a week, a total of 3 doses. Table 15. Study #3: Control and test treatment groups in CT-26 tumor bearing mice. potion Dose (mg/kg) way, plan number of mice Vehicle control + IgG isotype control 0 + 10 IV, QWx3 + IP, BIWx3 14 Compound A 1 IV, QWx3 14 Compound A 3 IV, QWx3 14 Compound A 6 IV, QWx3 14 Compound A 9 IV, QWx3 14 anti-PD-1 antibody 10 IP, BIWx3 14 Compound A+anti-PD-1 antibody 1 + 10 IV, QWx3 + IP, BIWx3 14 Compound A+anti-PD-1 antibody 3 + 10 IV, QWx3 + IP, BIWx3 14 Compound A+anti-PD-1 antibody 6 + 10 IV, QWx3 + IP, BIWx3 14 BIWx3=twice a week for 3 weeks, a total of 6 doses; IP=intraperitoneal; IV=intravenous; QWx3=once a week, a total of 3 doses; Q2Wx2=every 2 weeks, a total of 2 doses.

In Study 1, vehicle was administered IV once weekly for ³ doses (QWx3) or 0.3, 1, or 3 mg/ CT-26 tumor-bearing mice were treated with Compound A kg IV once a week for three doses (QWx3) or every 2 weeks for 2 doses (Q2Wx2).

In Study 2, CT-26 tumor-bearing mice were treated on day 5 after tumor cell inoculation, when the mean tumor volume was approximately 80 mm ³ . With Vehicle IV QWx3 + IgG isotype control IP, or Compound A according to QWx3 dosing schedule 3 or 6 mg/k IV, or anti-PD-1 antibody at 10 mg/kg IP, or Compound A at 6 mg/kg IV QWx3 Combination administration of A+10 mg/kg IP anti-PD-1 antibody. In all cases, IP administration of the antibody was twice weekly for 3 weeks for a total of 6 doses (BIWx3).

In Study 3, CT-26 tumor-bearing mice were treated on day 7 after tumor cell inoculation, when the mean tumor volume was approximately 70 mm ³ . Vehicle IV QWx3 + IgG isotype control IP BIWx3; or Compound A at 1, 3, 6, or 9 mg/kg IV according to the QWx3 dosing schedule, or anti-PD-1 antibody at 10 mg/kg IP BIWx3; or 1, Combination administration of 3 or 6 mg/kg IV QWx3 of compound A + 10 mg/kg IP BIWx3 of anti-PD-1 antibody.

A summary of all three studies is shown in Table 16. Animals were observed daily for clinical signs. Animals were humanely euthanized when tumor volumes grew beyond 2000 mm or they ^were observed to have a progressively worsening condition or show clear signs of severe distress and/or pain, according to IACUC guidelines.

Survival of each mouse was monitored over 100 days, at which point surviving tumor-free animals from Studies 2 and 3 were included in the rechallenge continuation of the study for two cycles, 2 months apart. Specifically, tumor-free animals were rechallenged by inoculating the same type of tumor cells (CT-26) in the opposite flank. Control animals were age-matched naive mice inoculated in parallel with the same number of CT-26 tumor cells on opposite flanks.

Tumor growth was monitored using digital caliper measurements every 3-4 days until the end of the study. Tumor volume was calculated as width2 x length/ ² , where width is the smallest dimension and length is the largest dimension. Raw tumor volume data are provided in the study report.

Mean tumor volume data for each group were plotted over time with standard error of the mean (SEM) bars. In addition, individual tumor volume data for the last day before animal sacrifice were plotted together with mean and SEM bars to examine the distribution of data.

Statistical analysis of tumor volume data on the last day before animal sacrifice was performed using GraphPad Prism v.7.0. Data were analyzed for significance using one-way ANOVA. Pairwise comparisons were performed using the Tukey test procedure (two-sided). p-values are reported for each individual comparison.

The percent tumor growth inhibition (TGI%) for each treatment group versus the control group was calculated as follows: [(Control-Control Baseline) - (Treatment-Treatment Baseline)]/(Control-Control Baseline) x 100%.

Survival of each mouse was recorded and Kaplan-Meier plots were generated to show survival by treatment group and significance was assessed by log-rank (Mantel-Cox) test. In Studies #1, #2, and #3, survival was monitored over 100 days after initiation of treatment, and in Studies #2 and #3, over two rechallenge cycles in surviving tumor-free mice. Analysis was performed using GraphPad Prism version 7.0. Table 16. Tumor growth inhibition of Compound A in mice as monotherapy and in combination with anti-mouse PD-1 antibody. Compound A dose [antibody dose ^a ] (mg/kg) TGI% (relative to vehicle control) Study #1 Study #2 Study #3 QWx3 Q2Wx2 QWx3 QWx3 Compound A Monotherapy 0.3 19 20 - - 1 31 27 - 30 3 51* 45* 56 59*** 6 - - 36* 86*** 9 - - - 85*** Antibody [10] - - 44 ^# 44 ^# Combination ^therapyb 6 + [10] - - 75** 84** ^a BIW was administered for 3 weeks (6 doses in total); ^b The data of the 1 and 3 mg/kg compound A combination groups are not shown. TGI% was calculated on Day 15 (Study 1) and Day 17 (Studies 2 and 3). Results are mean ± SEM. QWx3 = weekly for 3 doses; Q2Wx2 = every 2 weeks for 2 doses; TGI = tumor growth inhibition. *p < 0.05 vs. vehicle control; **p < 0.05 vs. monotherapy (compound A or antibody); ***p < 0.001 vs. vehicle or antibody isotype control; ^# vs. p < 0.01 compared to antibody isotype control.

In Study 1, the dose-dependent efficacy of Compound A as a single agent in female Balb/c mice bearing subcutaneously established CT-26 colon tumors was evaluated. When several tumor volumes in the control group exceeded 2000 mm, the study was officially terminated on day ¹⁵ after the start of treatment, according to the humane endpoint proposed by IACUC. Figure 26 shows the mean tumor volume over time for groups treated with Compound A at QWx3 dosing. Figure 27 shows the tumor volume at day 15 post-treatment for each animal treated with Compound A at QWx3 doses. Figure 28 shows the mean tumor volume over time for groups treated with Compound A at Q2Wx2 dosing. Figure 29 shows the tumor volume of each animal dosed Q2Wx2 with Compound A at day 15 post treatment.

According to the QWx3 dosing regimen, compound A exhibited dose-dependent single-agent antitumor activity, and the TGI% of the 0.3, 1 and 3 mg/kg dose groups compared with the vehicle control were 31%, 19% and 52%, respectively. Similarly, Compound A exhibited dose-dependent single-agent antitumor activity according to the Q2Wx2 dosing regimen, resulting in TGI% of 20%, 27% and 45% for the 0.3, 1 and 3 mg/kg dose groups compared to the vehicle control, respectively. %. However, only the 3 mg/kg dose was statistically significant (p < 0.05) compared to the vehicle control according to both dosing regimens. Both dosing regimens exhibited comparable antitumor activity. Therefore, for subsequent studies in this mouse model, the QWx3 dosing regimen was chosen.

In Figure 26 , Figure 28 , Figure 30 and Figure 33 , the black arrows indicate the days on which Compound A was administered. The data in Figure 26 and Figure 28 are mean tumor growth curves for QWx3 and Q2Wx2 administration of Compound A; black arrows indicate days of Compound A administration. The data in Figures 27 and 29 represent individual tumor volumes and mean tumor volume ± standard error of the mean (SEM) on day 15 following treatment with Compound A at QWx3 and Q2Wx2 doses (10 mice/group). Data represent individual tumor volumes; mean ± SEM and % TGI compared to vehicle control are also shown.

The data in Figure 28 represent mean tumor volume ± standard error of the mean (SEM) in animals dosed Q2Wx2 with Compound A (10 mice/group). The data in Figure 29 represent individual and mean tumor volume data at day 15 following treatment with Compound A administered at Q2Wx2. *p < 0.05 vs vehicle control at day 15.

Two separate studies (Studies 2 and 3) were performed in CT-26 colon tumor-bearing mice to evaluate Compound A as a single agent and in combination with a murine anti-PD-1 checkpoint inhibitor antibody. The dose range of Compound A overlapped between the studies, with Study 3 having a wider dose range. In both studies, Compound A was administered QWx3 and the same dose level of antibody was administered BIWx3.

In Study 2, the antitumor activity of Compound A at 3 and 6 mg/kg (QWx3) as a single agent was assessed in female Balb/c mice bearing subcutaneously established CT-26 colon tumors. In addition, compound A (QWx3) at 6 mg/kg and anti-PD-1 antibody (BIWx3) at 10 mg/kg IP were administered IV to evaluate combined antitumor activity. TGI% was calculated on day 15 after treatment initiation, as several tumors in the vehicle control group exceeded 2000 mm ³ in volume. However, animals in the treatment groups that exhibited complete tumor regression were followed for tumor measurement once or twice a week.

Compound A exhibited single-agent antitumor activity, resulting in TGI% of 56.3% and 35.6% for the 3 and 6 mg/kg dose groups compared to the vehicle control, respectively. In the combination study, treatment with Compound A IV at 6 mg/kg QWx3 starting 5 days after tumor cell inoculation, when the mean tumor volume was approximately 80 ^mm3 , or IP with anti-PD-1 antibody BIWx3, or as per Mice bearing CT-26 tumors were treated with the combination with the same dosing regimen. Figure 30 shows the mean mean values of mice treated with vehicle, 6 mg/kg Compound A as a single agent, anti-PD-1 antibody as a single agent, and a combination of 6 mg/kg Compound A and anti-PD-1 antibody. Tumor growth curve. Data in Figure 30 represent mean tumor volume ± SEM (14 mice/group). The upper arrows indicate the days of compound A administration, while the lower arrows indicate the days of anti-PD-1 antibody administration. Compared with compound A or anti-PD-1 antibody alone, the combined anti-tumor activity was significantly enhanced (p < 0.05). The TGI% profile is shown in Figure 31 and compared to the groups treated with vehicle, Compound A alone, or anti-PD-1 antibody alone, the group treated with the combination of Compound A and anti-PD-1 antibody was significantly more active on treatment. After 15 days showed a significant anti-tumor effect (35.6% for the compound A alone group; 44.1% for the anti-PD-1 antibody alone group; and 74.6% for the group administered the combination of compound A and anti-PD-1 antibody %). Data represent individual tumor volumes; mean ± SEM and % TGI compared to vehicle control are also shown. *p < 0.05, **p < 0.01, and ***p < 0.01 compared to vehicle control. ^┴ p < 0.05 compared with anti-PD-1 antibody. #p < 0.05 compared to Compound A. The median survival times for each group are shown in Figure 32 , and were 17, 27, 27.5, and 38 days for the control, Compound A, anti-PD-1 antibody, and Compound A+anti-PD-1 antibody groups, respectively. The median survival time of the combination group was significantly longer than that of compound A (p < 0.05) and anti-PD-1 antibody (p < 0.05) single-agent treatment groups. At 98 days after treatment, only 1 of 14 animals (7%) in each of the compound A and anti-PD-1 antibody dose groups survived tumor-free, compared with 4 of 14 animals (29%) in the combination group survive. The data in Figure 32 represent the Kaplan-Meier survival curves for the treatment groups. *p < 0.05 compared to vehicle control. ^┴ p < 0.05 compared with anti-PD-1 antibody. #p < 0.05 compared to compound A.

In Study 3, compared with Study 2, SC CT-26 colon tumor-bearing female Balb/c mice were dosed at a wider dose range (1, 3, 6 and 9 mg) according to the same IV QWx3 dosing schedule. /kg) to evaluate the single-agent antitumor activity of compound A. The data in Figure 33 represent mean tumor growth curves for Compound A administered at 1 mg/kg, 3 mg/kg, 6 mg/kg, and 9 mg/kg as a single agent. Data represent mean tumor volume ± SEM (14 mice/group; except 9 mg/kg Compound A which was 12 mice/group). Black arrows indicate the days on which Compound A was administered. Compound A administered alone at 1 mg/kg, 3 mg/kg, 6 mg/kg and 9 mg/kg also exhibited dose-dependent antitumor activity, as compared with the vehicle control, 1, 3, 6 and The TGI% of the 9 mg/kg dose group were 29.8%, 58.8%, 86.2% and 84.8%, respectively ( Figure 34 ). TGI% was calculated on day 15 after treatment initiation, as several tumors in the vehicle control group exceeded 2000 mm ³ . However, animals in the treatment groups that exhibited complete tumor regression were followed for tumor measurement once or twice a week. The data in Figure 34 represent individual tumor volumes at day 15 post-treatment. Data represent individual tumor volumes; mean ± SEM and % TGI compared to vehicle control are also shown. ***p < 0.01 vs vehicle control. The lowest dose (1 mg/kg) showed no statistically significant antitumor activity compared to the vehicle-treated group, whereas the other 3 dose groups were statistically significant (p < 0.001). The data also showed that the TGI% was similar in the two high dose groups (6 mg/kg and 9 mg/kg), indicating that maximal antitumor activity was achieved at the 6 mg/kg dose. In the 9 mg/kg dose group, 2 of 14 animals were found to die after losing >15% body weight due to treatment.

In the combination phase of the study, Compound A (QWx3) at 1, 3 or 6 mg/kg was administered together with anti-PD-1 antibody (BIWx3) at 10 mg/kg IP. Treatment with Compound A IV at 1, 3, 6, or 9 mg/kg QWx3 starting 7 days after tumor cell inoculation, when the mean tumor volume was approximately 70 mm3, or IP with anti-PD-1 antibody BIWx3, or CT-26 tumor-bearing mice were treated with the combination following the same dosing schedule. Note that for the 9 mg/kg Compound A single agent group, two animals were found to die after >15% body weight loss and were not included in the analysis. With the combination of 1 mg/kg Compound A + anti-PD-1 antibody, no additive antitumor activity was observed based on survival data. At days after Compound A treatment, 1 of 14 animals (7%) survived in the anti-PD-1 antibody group compared to 0 animals in the 3 mg/kg single agent group. However, in the 3 mg/kg Compound A + anti-PD-1 antibody group, 2 of 14 animals (14%) survived until Compound A days. As shown in Figure 35 , the combination of 6 mg/kg Compound A + anti-PD-1 antibody resulted in prolonged survival compared to each single agent alone. Vehicle control, compound A (6 mg/kg), anti-PD-1 antibody (10 mg/kg), and compound A+anti-PD-1 antibody group (6 mg/kg compound A and 10 mg/kg anti-PD-1 antibody) had median survival times of 21, 35, 24.5, and 49 days, respectively. The median survival time of the combination group was significantly longer than that of compound A and anti-PD-1 antibody (p<0.05) single agent treatment group. Specifically, 0 animals in the 6 mg/kg Compound A group survived days after Compound A treatment, while only 1 (7%) of 14 animals in the anti-PD-1 antibody group survived without tumors. However, in the combination group, 5 of 14 animals (36%) survived tumor-free (p < 0.05). The data in Figure 35 represent the Kaplan-Meier survival curves for the treatment groups. *p < 0.05 compared to vehicle control. ┴ p < 0.05 compared with anti-PD-1 antibody. #p < 0.05 compared to Compound A. Example 6. PK/PD studies in naive ( E3826-U1704 ) and B16-F10 tumor bearing ( E3826-U1803 ) C57BL/6 mice .

This study describes the effect of certain IL-2 conjugates as described herein in a murine melanoma model. The study design is summarized in Tables 17 and 18, where doses were calculated by reference to the mass of the protein component excluding the mass of the PEG moiety. Terminal blood samples were collected by cardiac puncture at the indicated points. Study E3826-U1704 included 13 time points (0.13, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 and 120 h), 3 mice were sacrificed at each time point, and the study E3826-U1803 included 9 time points (2, 8, 12, 24, 48, 72, 120, 168 and 240 h), and 4-7 mice were sacrificed at each time point. Plasma and blood cells (in both studies) and tumors in Study E3826-U1803 were collected for PK and PD analysis.

IL-2 conjugates P65_30kD and E62_30kD were used in this study. IL-2 conjugate P65_30kD refers to the conjugate "IL-2_P65[AzK_PEG30kD]" described in Example 5. IL-2接合物E62_30kD包含SEQ ID NO: 5： APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG30kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 5) 其中[AzK_ PEG30kD]是經由DBCO介導的點擊化學與PEG穩定接合的N6-((2-疊Nitroethoxy)-carbonyl)-L-lysine, said click chemistry forms a compound comprising a structure of formula (I), wherein Z, Y, W and q are as in Example 5 for "IL-2_P65[AzK_PEG30kD ]” said. The IL-2 conjugate E62_30kD was prepared using a method in which a protein with SEQ ID NO: 4 was first prepared, wherein the glutamic acid at position 62 was replaced by N 6-((2-azidoethoxy)-carbonyl)-L- Lysine AzK substitution. The AzK-containing protein was then allowed to react under click chemistry conditions with DBCO containing a methoxyl linear PEG group with an average molecular weight of 30 kDa, followed by purification and formulation using standard procedures.

Bioanalysis of plasma samples was performed using a qualified human IL-2 ELISA assay (Abcam, Cambridge, UK). ELISA assays were used to determine the concentrations of aldesleukins, E62_30kD and P65_30kD and internal standards in plasma derived samples. PK data analysis was performed at NW Solutions (Seattle, WA). PK data were imported into Phoenix WinNonlin v6.4 (Certara/Pharsight, Princeton, NJ) for analysis. The group mean plasma concentration versus time data were analyzed using the IV bolus administration model in a non-compartmental approach. Table 17 PK/PD Study No. E3826-U1704 - Control and Test Treatment Groups in Naive C57/BL6 Mice. treat Dose *(mg/Kg) way, plan point in time N control 0 IV, single dose 13 3 aldesleukin 0.3 IV, single dose 13 3 P65_30kD 0.3 IV, single dose 13 3 E62_30kD 0.3 IV, single dose 13 3 *Dose refers to the amount of IL-2 polypeptide, where the dose is calculated by reference to the mass of the protein component excluding the mass of the PEG moiety. Table 18 PK/PD Study No. E3826-U1803 - Control and Test Treatment Groups in B16F-10 Melanoma Tumor Bearing Mice. treat Dose *(mg/kg) way, program point in time N None (before administration) 0 mg/kg none 1 6 Vehicle control 0 mg/kg IV, single dose 9 3 P65_30kD 1 mg/kg IV, single dose 9 4 P65_30kD 3mg/kg IV, single dose 9 4 *Dose refers to the amount of IL-2 polypeptide, where the dose is calculated by reference to the mass of the protein component excluding the mass of the PEG moiety.

In study E3826-U1704, both P65_30kD and E62_30kD exhibited a superior PK profile relative to aldesleukin, as summarized in Table 19. Following a single IV bolus dose of aldesleukin, Tmax was observed at 0.03 h post-dose (first measured time point post-dose), and mean plasma concentrations were measurable up to 4 h post-dose. Following single IV bolus administration of P65_30kD and E62_30kD, Tmax was observed at 0.03 h post-dose and mean plasma concentrations were measurable until 120 h post-dose (the last measurement time point). In a separate study, following IV administration of E62_5kD (an IL-2 conjugate similar to E62_30kD but with a PEG group with an average molecular weight of 5 kDa), _Tmax was observed at 0.133 hr post-dose and until 12 hr post-dose, Mean plasma concentrations are measurable.

Exposure based on C _max and AUC _0-t was as follows: P65_30kD > E62_30kD >> E62_5kD > aldesleukin. The PK profile of E62_5kD with smaller PEG was closer to that of rIL-2 (Table 15). Based on Cmax and AUC0-t, P65_30kD exposure was 5.5-fold and 200-fold higher than aldesleukin, respectively. In addition, P65_30kD showed a 23-fold prolonged t1/2 (13.3 h vs. 0.57 h) and an approximately 198-fold decrease in CL (6.58 vs. 1300 mL/h/Kg) compared to aldesleukin. For both P65_30kD and E62_30kD, the volume of distribution (82.4 mL/Kg and 92.3 mL/Kg, respectively) was reduced by about 4.2 to 4.7-fold relative to aldesleukin and was similar to the blood volume in mice (85 mL/Kg Kg; [Boersen 2013]). This indicated that P65_30kD and E62_30kD were most distributed in systemic circulation. Table 19. P65_30kD PK parameters in C57BL/6 female mice. parameter unit P65_30kD E62_30kD E62_5kD aldesleukin _Tmax h 0.030 0.030 0.133 0.030 _Cmax ng/mL 4,870 4,230 936 884 AUC _0-t h*ng/mL 45,600 37,100 798 229 R ² 0.992 0.986 0.851 0.900 AUC _INF h*ng/mL 45,600 37,100 807 230 t _1/2 h 13.300 14.500 2.56 0.573 CL mL/h/Kg 6.580 8.07 372 1300 V _SS mL/Kg 82.4 92.3 404 390 NOTE: R2 is the goodness ^- of-fit parameter at the end of each concentration versus time curve. All parameters display 3 significant figures. Example 7. Pharmacodynamic studies of IL-2 conjugates. Pharmacodynamic observations in the peripheral blood compartment.

STAT5 phosphorylation and induction of cell proliferation (early molecular marker Ki-67 and cell count) were used as pharmacodynamic readouts to assess the pharmacological profile of P65_30kD described in Example 4 relative to its pharmacokinetics. In CD8+ effector T cells, the pSTAT5 PD marker showed good correlation with the PK of both P65_30kD and aldesleukin (Table 13). Sustained elevation of pSTAT5 was observed for up to 72 hours in NK and CD8+ T cells and up to 24 hours in Tregs. In aldesleukin-administered mice, pSTAT5 induction returned to baseline after only 2 h. Phosphorylation of STAT5 switched to a proliferative response (72–120 h) in CD8+ effector T cells and NK cells, but not T regs, and phenotypic analysis of CD8+ effector T cells revealed a marked expansion of CD44+ memory cells within this population.   Pharmacodynamic observations in the tumor compartment of B16-F10 tumor-bearing ( E3826-U1803 ) C57BL/6 mice.

Table 20 shows plasma and tumor drug concentrations following a single dose of 3 mg/kg of P65_30kD in B16-F10 tumor-bearing mice, where doses were calculated by referring to the mass of the protein component excluding the mass of the PEG moiety . The tumor half-life was twice that of the plasma half-life (24.4 vs. 12.6), suggesting that P65_30kD penetrated and remained in the tumor. The tails of the curves cross, showing faster plasma elimination than tumor (data not shown). Tumor:plasma AUC ratios were 9.7% and 8.4% for the 1 mg/kg and 3 mg/kg doses, respectively. Table 20 P65_30kD plasma and tumor PK parameters of B16-F10 tumor-bearing C57BL/6 female mice. parameter P65_30kD (3 mg/kg) plasma the tumor T _max (h) *2.00 8 C _max (ng/mL) 40000 1550 t1/2(h) 12.60 24.4 AUC _0-t (h*ng/mL) 656,000 55200 R ² 0.974 0.988 AUC _INF (h*ng/mL) 656,000 55200   MTD study in Balb/c mouse E3826- U1802

A dose-ranging study of P65_30kD was performed in naive female Balb/c mice at Crown Biosciences, Inc. (San Diego, CA). The study design is shown in Table 21 in which doses were calculated by referring to the mass of the protein component excluding the mass of the PEG moiety. Blood samples were drawn via the submandibular vein at 8 time points (0.25, 1, 4, 12, 24, 34, 48, and 72 h). Both plasma and blood cells were collected for PK and PD analysis.

All plasma samples were analyzed for human IL-2 and mouse IL-2, TNF-α, IFNγ, IL-5 and IL-6 cytokines using commercially available ELISA kits. Table 21 PK/PD and MTD Study No. E3826-U1802 - Control and Test Treatment Groups in Naive Balb/C Mice. treat Dose (mg/kg) way, program point in time N initial test 0 mg/kg 0 3 Vehicle control 0 mg/kg IV, BID x 3 3 3 aldesleukin 0.01 mg/kg IV, BID x 3 3 3 aldesleukin 0.03 mg/kg IV, BID x 3 3 3 aldesleukin 0.1 mg/kg IV, BID x 3 3 3 aldesleukin 1.0 mg/kg IV, BID x 3 3 3 aldesleukin 3.0 mg/kg IV, BID x 3 3 3 aldesleukin 5.0 mg/kg IV, BID x 3 3 3 P65_30kD 0.01 mg/kg IV, single dose 3 3 P65_30kD 0.03 mg/kg IV, single dose 3 3 P65_30kD 0.1 mg/kg IV, single dose 3 3 P65_30kD 1.0 mg/kg IV, single dose 3 3 P65_30kD 3.0 mg/kg IV, single dose 3 3 P65_30kD 5.0 mg/kg IV, single dose 3 3 #P65_30kD 0.3 mg/kg IV, single dose 8 3 * Blood was collected via the submandibular vein at all time points except the 72 hr time point. Peripheral blood was collected at the 72 hr time point. # Only P65_30kD at 0.3 mg/kg was used for PK/PD evaluation.   Toxicological observations in the MTD study using Balb/c mice

The major toxicities associated with high doses of aldesleukin are vascular leak syndrome and the related cytokine release syndrome (CRS). To assess the potential of this effect in mice, a single dose IV P65_30kD administration (Table 21 ) was performed at doses ranging from 0.01-5.0 mg/kg by reference to the protein fraction excluding the mass of the PEG moiety mass to calculate the dose. The analyzes performed were hematology, histopathology, organ weight and cytokine analysis. No abnormalities were observed for hematology, histopathology or body weight relative to vehicle control mice with P65_30kD or aldesleukin. Regarding cytokine analysis, aldesleukin was observed to increase plasma IL-5 levels starting at 1 mg/kg to 5 mg/kg. With P65_30kD, only a moderate increase in IL-5 was observed at the 5 mg/kg dose (but low compared to aldesleukin). A transient increase in systemic levels of IFNγ was observed with both aldesleukin and P65_30kD. Example 8. Clinical Study of Combination Therapy Using IL-2 Conjugates and Cimiprizumab.

A Phase 1/2 nonrandomized, open-label, multigroup, multicenter study was conducted to evaluate the IL-2 conjugate described in Example 1 in combination with cimiprimumab for the treatment of patients with advanced unresectable or Clinical benefit for participants with metastatic skin cancer. Cohort A participants were immune checkpoint inhibitor (ICI)-naive patients with locally advanced, unresectable, or metastatic melanoma who had not received prior therapy (i.e., IL-2 conjugate therapy was 1L or first-line therapy ; subjects were treatment-naïve). Cohort B participants were immune checkpoint inhibitor (ICI)-naïve patients with metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who were not candidates for curative surgery or curative radiation and had received prior More than 2 prior lines of systemic therapy (ie, IL-2 conjugate therapy was 1-3L, or first to third line therapy).

Participants in cohorts A and B received IL-2 conjugates (16 or 24 μg/kg dose) and simiprizumab (350 mg) every 3 weeks (day 1 of each cycle). Participants were administered acetaminophen, diphenhydramine, and ondansetron (or equivalent) prior to the first 4 cycles of treatment. For subsequent cycles, prior to treatment, the supervising physician may have decided to administer acetaminophen, diphenhydramine, and/or ondansetron if determined to be medically necessary or appropriate. Repeat treatment for a total of up to 35 cycles (1 cycle is 21 days).

Key inclusion criteria. All participants were male or female over the age of 18. Participants in Cohort A had a histologically confirmed diagnosis of unresectable locally advanced or metastatic melanoma not amenable to local therapy. Participants in cohort B had a histologically confirmed diagnosis of locally advanced or metastatic cutaneous squamous cell carcinoma (CSCC), had received no more than 2 prior lines of systemic therapy, and were not candidates for curative surgery or radiation. Participants in cohort A had at least one measurable lesion according to RECIST 1.1 criteria. Participants in cohort B had at least one lesion measurable according to RECIST 1.1, or at least one lesion that could be followed by serial digital medical photographs using modified WHO criteria. All participants had adequate cardiovascular, hematological, hepatic, renal function and laboratory parameters.

Key exclusion criteria. Participants had no history of allogeneic tissue or solid organ transplantation. Participants had no grade 4 immune-mediated/related toxicities from prior immuno-oncology therapy, or immune-mediated/related toxicities leading to discontinuation. Participants did not have any grade ≥ 2 persistent AEs attributable to prior anticancer therapy. Participants did not have a baseline oxygen saturation (SpO2) of ≤ 92% (without oxygen therapy). Participants were allowed to temporarily (at least 36 hours) withhold antihypertensive medications prior to each IL-2 conjugate administration. Participants were free of any medical or clinical condition, laboratory abnormality, or any specific condition that, in the judgment of the supervising physician, might preclude protocol therapy or might render the subject unsuitable for the study. Participants in Cohort A did not have uveal or ocular melanoma or desmoplastic melanoma. Participants in Cohort B did not have vermilion (vermilion) or anogenital area as the primary site of CSCC and mixed CSCC histology (eg, sarcomatoid, adenosquamous). All participants had an ECOG performance status of less than 2.

Patients were monitored for disease progression according to various criteria. Evaluation according to RECIST 1.1, using modified WHO criteria or combination, optionally up to the date of first documented progression, in patients following administration of the first dose of combination therapy with IL-2 conjugate and cimiprizumab Objective response rate (ORR). According to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0 and the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading, from signing the informed consent form (ICF) until 90 days after the last administration of study treatment or until participation Patients started another anticancer therapy (whichever was earlier), and evaluated the incidence of treatment-emergent adverse events (TEAEs), dose-limiting toxicities (DLTs), serious adverse events (SAEs) and laboratory abnormalities. Complete response rate (CRR) and time to CR were evaluated per RECIST 1.1 for Cohort A participants and, if applicable, Cohort B participants until the first dose of IL-2 conjugate and cimipril was administered About 6 months after monoclonal antibody combination treatment. Based on the supervising physician's assessment, the timing of the evaluation of the above parameters was adjusted as needed. For example, any of the variables described above can be assessed up to 36 months after administration of the first therapeutic dose.

The following parameters were also evaluated in patients up to approximately 36 months after administration of the first dose of the IL-2 conjugate and simiprizumab combination therapy: (1) Time to Response (TTR), according to RECIST 1.1, modified (2) Duration of response (DoR), defined as from first documented evidence of CR or PR to disease progression (PD) (according to RECIST 1.1, modified WHO criteria for medical photographs or combined criteria criteria) or death from any cause (whichever occurs first); (3) clinical benefit rate (CBR), including those confirmed at any time according to RECIST 1.1, modified WHO criteria for medical photographs, or a combination of criteria CR or PR plus stable disease (SD) for at least 6 months; and (4) progression-free survival (PFS), defined as the period from the first administration of the IL-2 conjugate and cimiprizumab combination Date of treatment to date of first documented disease progression (according to RECIST 1.1 or modified WHO criteria for medical photographs) or death from any cause, whichever occurred first. Pharmacokinetic parameters, such as the concentration of IL-2 conjugates, the incidence of anti-drug antibodies (ADA) against IL-2 conjugates, C _trough and C _{infusion of simiprizumab ends} .

Number of participants with treatment-emergent adverse events (TEAEs) by major system organ class (SOC) and participant (PT) worst grade in Cohort A (5 patients) and Cohort B (4 patients) ( %) are detailed in Table 22. Table 22 Treatment Emerging Adverse Events ( TEAEs ) Cohort A IL-2 conjugate (16 ug/kg) + cimiprizumab (N=5) Cohort B IL-2 conjugate (16 ug/kg) + cimiprizumab (N=4) All (N=9) Major system organ class preference, n (%) all grades Grading≥3 _ all grades Grading≥3 _ all grades Grading≥3 _ any event 5 (100) 2 (40.0) 3 (75.0) 2 (50.0) 8 (88.9) 4 (44.4) Blood and Lymphatic System Disorders 2 (40.0) 1 (20.0) 1 (25.0) 1 (25.0) 3 (33.3) 2 (22.2) Thrombocytopenia 2 (40.0) 1 (20.0) 1 (25.0) 0 3 (33.3) 1 (11.1) hypereosinophilia 0 0 1 (25.0) 1 (25.0) 1 (11.1) 1 (11.1) immune system disorder 2 (40.0) 0 0 0 2 (22.2) 0 cytokine release syndrome 2 (40.0) 0 0 0 2 (22.2) 0 Metabolic and Nutritional Disorders 0 0 1 (25.0) 1 (25.0) 1 (11.1) 1 (11.1) hyperglycemia 0 0 1 (25.0) 1 (25.0) 1 (11.1) 1 (11.1) mental disorder 0 0 1 (25.0) 0 1 (11.1) 0 anxiety 0 0 1 (25.0) 0 1 (11.1) 0 nervous system disorder 1 (20.0) 0 1 (25.0) 0 2 (22.2) 0 Headache 1 (20.0) 0 1 (25.0) 0 2 (22.2) 0 Eye disorders 0 0 1 (25.0) 0 1 (11.1) 0 Diplopia 0 0 1 (25.0) 0 1 (11.1) 0 heart disorder 1 (20.0) 1 (20.0) 0 0 1 (11.1) 1 (11.1) Myocarditis 1 (20.0) 1 (20.0) 0 0 1 (11.1) 1 (11.1) Vascular disorders 0 0 1 (25.0) 0 1 (11.1) 0 hypertension 0 0 1 (25.0) 0 1 (11.1) 0 Gastrointestinal disorders 1 (20.0) 0 0 0 1 (11.1) 0 Vomit 1 (20.0) 0 0 0 1 (11.1) 0 Skin and Subcutaneous Tissue Disorders 1 (20.0) 0 2 (50.0) 0 3 (33.3) 0 dermatitis 0 0 2 (50.0) 0 2 (22.2) 0 sweating 1 (20.0) 0 0 0 1 (11.1) 0 rash 1 (20.0) 0 0 0 1 (11.1) 0 Maculopapular rash 0 0 1 (25.0) 0 1 (11.1) 0 Musculoskeletal and connective tissue disorders 2 (40.0) 1 (20.0) 1 (25.0) 0 3 (33.3) 1 (11.1) joint pain 0 0 1 (25.0) 0 1 (11.1) 0 Myalgia 1 (20.0) 0 0 0 1 (11.1) 0 myositis 1 (20.0) 1 (20.0) 0 0 1 (11.1) 1 (11.1) trismus 0 0 1 (25.0) 0 1 (11.1) 0 General disorders and the status of the administration site 3 (60.0) 0 1 (25.0) 0 4 (44.4) 0 fatigue 2 (40.0) 0 1 (25.0) 0 3 (33.3) 0 fever 2 (40.0) 0 0 0 2 (22.2) 0 an examination 1 (20.0) 1 (20.0) 2 (50.0) 2 (50.0) 3 (33.3) 3 (33.3) Elevated alanine transaminase 1 (20.0) 0 2 (50.0) 2 (50.0) 3 (33.3) 2 (22.2) Elevated aspartate aminotransferase 1 (20.0) 0 2 (50.0) 1 (25.0) 3 (33.3) 1 (11.1) Elevated blood bilirubin 1 (20.0) 1 (20.0) 0 0 1 (11.1) 1 (11.1) elevated body temperature 0 0 1 (25.0) 0 1 (11.1) 0 Injury, poisoning and surgical complications 1 (20.0) 0 2 (50.0) 0 3 (33.3) 0 infusion related reactions 1 (20.0) 0 2 (50.0) 0 3 (33.3) 0 MedDRA 24.1, NCI CTCAE Version 5.0 n (%) = number and percentage of participants with at least one TEAE The table is sorted by the order of SOC international recognition and the decreasing frequency of PT.

Four individuals with CSCC (cohort B) received a dose of 16 μg/kg Q3W of IL-2 conjugate in combination with simiprizumab (350 mg Q3W). For 3 of 4 individuals, the investigator reported an unconfirmed response (ie, significant reduction in target lesion size) after the first tumor assessment; for these 3 individuals, the first tumor assessment occurred in cycle 3 Rear. For the remaining individuals, investigators reported confirmed pseudoprogressions during the first cycle, followed by unconfirmed responses (i.e., marked reduction in size of one or more target lesions) after 7 weeks.

Five individuals with melanoma received a dose of 16 μg/kg Q3W of an IL-2 conjugate in combination with simiprizumab (350 mg Q3W). In some embodiments, the individual exhibits a reduction in the size of one or more target lesions after one treatment cycle. In some embodiments, the individual exhibits a decrease in the size of one or more target lesions after the first tumor assessment. In some embodiments, the individual demonstrates a response (ie, a decrease in the size of the target lesion) after the second, third, or fourth tumor assessment. In some embodiments, the subject exhibits a response (ie, a decrease in the size of the target lesion) after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles. In some embodiments, the subject exhibits a response (i.e., target lesion size decreases).

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that these embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

none

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description and accompanying drawings which illustrate illustrative embodiments utilizing the principles of the invention, in which:

Figure 1A shows changes in peripheral CD8+ T _eff counts in indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after IL-2 conjugate administration. Here and elsewhere, names such as "C1D1" denote treatment cycles and days (eg, treatment cycle 1, day 1). "Pre (PRE)" means a baseline measurement before administration; 24HR means 24 hours after administration; and so on.

Figure 1B shows the peak expansion of peripheral CD8+ T _eff cells following administration of the first dose of 24 μg/kg [Q3W] of IL-2 conjugates. Data were normalized to pre-treatment (C1D1) CD8+ T cell counts.

Figure 1C shows peripheral CD8+ T _eff cell counts in indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after IL-2 conjugate administration.

Figure 2 shows the percentage of Ki67 expressing CD8+ T _eff cells in the indicated subjects treated with 24 μg/kg [Q3W] of IL-2 conjugates at the indicated times after administration of IL-2 conjugates.

Figure 3A shows changes in CD8+ memory cell counts in indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates.

Figure 3B shows CD8+ memory cell counts in the indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after IL-2 conjugate administration.

Figure 4A shows changes in peripheral natural killer (NK) cell counts in indicated subjects treated with 24 μg/kg [Q3W] of IL-2 conjugates at the indicated times after administration of IL-2 conjugates.

Figure 4B shows the peak peripheral NK cell expansion following administration of the first dose of 24 μg/kg [Q3W] of IL-2 conjugates. Data were normalized to pre-treatment (C1D1) NK cell counts.

Figure 4C shows changes in peripheral natural killer (NK) cell counts in indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after IL-2 conjugate administration.

Figure 4D shows peripheral natural killer (NK) cell counts in the indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after IL-2 conjugate administration.

Figure 5 shows the percentage of Ki67 expressing NK cells in the indicated subjects treated with 24 μg/kg [Q3W] of IL-2 conjugate at the indicated times after IL-2 conjugate administration.

Figure 6A shows changes in peripheral CD4+ T _reg counts in indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after IL-2 conjugate administration.

Figure 6B shows the peak expansion of peripheral CD4+ T _reg cells following administration of the first dose of 24 μg/kg [Q3W] of IL-2 conjugates. Data were normalized to pre-treatment (C1D1) CD4+ T-cell counts.

Figure 6C shows peripheral CD4+ T _reg cell counts in indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after IL-2 conjugate administration.

Figure 7 shows the percentage of Ki67 expressing CD4+ T _reg cells in the indicated subjects treated with 24 μg/kg [Q3W] of IL-2 conjugates at the indicated times after IL-2 conjugate administration.

Figure 8A shows changes in eosinophil counts in the indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after administration of the IL-2 conjugates.

Figure 8B shows the peak peripheral eosinophil expansion following administration of the first dose of 24 μg/kg [Q3W] of IL-2 conjugates. Data were normalized to pre-treatment (C1D1) eosinophil counts.

Figure 8C shows eosinophil counts in the indicated subjects treated with IL-2 conjugates at 24 μg/kg [Q3W] at the indicated times after IL-2 conjugate administration.

Figure 9A shows the levels of IFN-γ, IL-5 and IL-6 in the indicated subjects treated with 24 μg/kg [Q3W] of IL-2 conjugates at the indicated times after administration of the IL-2 conjugates serum level.

Figure 9B shows serum levels of IL-5 following administration of 24 µg/kg [Q3W] of IL-2 conjugates. BLQ = lower limit of quantitation. Data are plotted as mean (range BLQ to max).

Figure 9C shows serum levels of IL-6 following administration of 24 µg/kg [Q3W] of IL-2 conjugates. BLQ = lower limit of quantitation. Data are plotted as mean (range BLQ to max).

Figure 10A shows the change in peripheral CD8+ T _eff counts in the indicated subjects at the indicated times after administration of 8 μg/kg IL-2 conjugate and pembrolizumab.

Figure 10B shows the change in peak expansion of peripheral CD8+ T _eff cells following administration of the first dose of IL-2 conjugate and pembrolizumab. Data were normalized to pre-treatment (C1D1) CD8+ T cell counts. Values listed represent median fold change.

Figure 10C shows the change in peripheral CD8+ T _eff counts in the indicated subjects at the indicated times after administration of 16 μg/kg IL-2 conjugate and pembrolizumab.

Figure 11 shows the percentage of CD8+ T _eff cells expressing Ki67 in the indicated subjects at the indicated times after administration of 8 μg/kg IL-2 conjugate and pembrolizumab.

Figure 12A shows the changes in peripheral natural killer (NK) cell counts in the indicated subjects at the indicated times after administration of 8 μg/kg IL-2 conjugate and pembrolizumab.

Figure 12B shows the change in peak peripheral NK cell expansion following administration of the first dose of IL-2 conjugate and pembrolizumab. Data were normalized to pre-treatment (C1D1) NK cell counts. Values listed represent median fold change.

Figure 12C shows the changes in peripheral natural killer (NK) cell counts in the indicated subjects at the indicated times after administration of 16 μg/kg IL-2 conjugate and pembrolizumab.

Figure 13 shows the percentage of NK cells expressing Ki67 in the indicated subjects at the indicated times after administration of 8 μg/kg IL-2 conjugate and pembrolizumab.

Figure 14A shows the change in peripheral CD4+ T _reg counts in the indicated subjects at the indicated times after administration of 8 μg/kg IL-2 conjugate and pembrolizumab.

Figure 14B shows the change in peak expansion of peripheral CD4+ T _reg cells following administration of the first dose of IL-2 conjugate and pembrolizumab. Data were normalized to pre-treatment (C1D1) CD4+ T-cell counts. Values listed represent median fold change.

Figure 14C shows the change in peripheral CD4+ T _reg counts in the indicated subjects at the indicated times after administration of 16 μg/kg IL-2 conjugate and pembrolizumab.

Figure 15 shows the percentage of CD4+ T _reg cells expressing Ki67 in the indicated subjects at the indicated times after administration of 8 μg/kg IL-2 conjugate and pembrolizumab.

Figure 16A shows the change in eosinophil counts in the indicated subjects at the indicated times after administration of 8 μg/kg IL-2 conjugate and pembrolizumab.

Figure 16B shows the change in peak peripheral eosinophil expansion following administration of the first dose of IL-2 conjugate and pembrolizumab. Data were normalized to pre-treatment (C1D1) eosinophil counts. Values listed represent median fold change.

Figure 16C shows the change in eosinophil counts in the indicated subjects at the indicated times after administration of 16 μg/kg IL-2 conjugate and pembrolizumab.

Figure 17A shows serum levels of IFN-γ, IL-5 and IL-6 in the indicated subjects at the indicated times after administration of 8 μg/kg IL-2 conjugate and pembrolizumab.

Figure 17B shows serum levels of IL-5 following administration of 8 μg/kg IL-2 conjugate and pembrolizumab. BLQ = lower limit of quantitation. Data are plotted as mean (range BLQ to max).

Figure 17C shows serum levels of IL-6 following administration of 8 μg/kg IL-2 conjugate and pembrolizumab. BLQ = lower limit of quantitation. Data are plotted as mean (range BLQ to max).

Figure 17D shows serum levels of IFN-γ, IL-5 and IL-6 in the indicated subjects at the indicated times after administration of 16 μg/kg IL-2 conjugate and pembrolizumab.

Figures 18A and 18B show mean concentrations of IL-2 conjugates administered with pembrolizumab at a dose of 8 μg/kg after 1 and 2 cycles, respectively.

Figure 18C and Figure 18D show the mean concentrations of IL-2 conjugates administered with pembrolizumab at a dose of 16 μg/kg after 1 and 2 cycles, respectively.

Figure 19 shows peripheral CD8+ T _eff cell counts in the indicated subjects at the indicated times after administration of 24 μg/kg IL-2 conjugate and pembrolizumab.

Figure 20 shows peripheral NK cell counts in the indicated subjects at the indicated times after administration of 24 μg/kg IL-2 conjugate and pembrolizumab.

Figure 21 shows the change in peripheral CD4+ T _reg cell counts in the indicated subjects at the indicated times after administration of 24 μg/kg IL-2 conjugate and pembrolizumab.

Figure 22 shows peripheral eosinophil counts in the indicated subjects at the indicated times after administration of 24 μg/kg IL-2 conjugate and pembrolizumab.

Figures 23A and 23B show mean concentrations of IL-2 conjugates administered with pembrolizumab at a dose of 24 μg/kg after 1 and 2 cycles, respectively .

Figure 24 shows IFN-γ, IL-6 and IL-5 in the indicated subjects treated with 24 μg/kg of IL-2 conjugate and pembrolizumab at the indicated times after administration of IL-2 conjugate level.

Figure 25A shows serum levels of IFN-γ, IL-5, and IL-6 in the indicated melanoma subjects at the indicated times following administration of IL-2 conjugates. BLQ = lower limit of quantitation.

Figure 25B shows the serum levels of IFN-γ, IL-5 and IL-6 in the indicated basal cell carcinoma (BCC) subjects at the indicated times after administration of IL-2 conjugates. BLQ = lower limit of quantitation.

Figure 26 shows a graph of the antitumor activity of Compound A administered according to QWx3 schedule IV in Study 1 in Example 5. Black arrows indicate the days of compound A dosing.

Figure 27 shows a graph of tumor volume in Study 1 in Example 5 with Compound A administered IV according to the QWx3 schedule.

Figure 28 shows the tumor volume at Day 15 post-treatment for each animal treated with Compound A at QWx3 doses in Study 1 in Example 5. Black arrows indicate the days of compound A dosing.

Figure 29 shows the tumor volume at day 15 post treatment for each animal dosed Q2Wx2 with Compound A in Study 1 in Example 5.

Figure 30 shows Example 5 study 2 with vehicle, 6 mg/kg Compound A as a single agent, anti-PD-1 antibody as a single agent, and combination treatment of 6 mg/kg Compound A with anti-PD-1 antibody Average tumor growth curves of mice. Black arrows indicate the days of compound A dosing.

Figure 31 shows the treatment in the group treated with the combination of Compound A and anti-PD-1 antibody compared with the group treated with vehicle, Compound A alone, or anti-PD-1 antibody alone in Study 2 of Example 5. Graph of TGI% data after 15 days. *p < 0.05, **p < 0.01, and ***p < 0.01 compared to vehicle control. ^┴ p < 0.05 compared with anti-PD-1 antibody. #p < 0.05 compared to compound A. Data represent mean tumor volume ± SEM (14 mice/group).

Figure 32 shows the Kaplan-Meier survival curves for the treatment groups in Study 2 of Example 5. *p < 0.05 compared to vehicle control. ^┴ p < 0.05 compared with anti-PD-1 antibody. #p < 0.05 compared to Compound A.

Figure 33 represents mean tumor growth curves for Compound A in Study 3 of Example 5 at 1 mg/kg, 3 mg/kg, 6 mg/kg, and 9 mg/kg single agent administration. Data represent mean tumor volume ± SEM (14 mice/group; 12 mice/group except Compound A at 9 mg/kg). Black arrows indicate the days on which Compound A was administered.

Figure 34 represents individual tumor volumes at Day 15 post-treatment in Study 3 of Example 5. Data represent individual tumor volumes; mean ± SEM and % TGI compared to vehicle control are also shown. ***p < 0.01 vs. vehicle control.

Figure 35 shows a graph of Kaplan-Meier survival curves for the treatment groups treated with vehicle (control), anti-PD-1 antibody alone, Compound A alone, and the combination of Compound A and anti-PD-1 antibody. *p < 0.05 in Study 3 of Example 5 vs. vehicle control. ^┴ p < 0.05 compared with anti-PD-1 antibody. #p < 0.05 compared to compound A.

Figure 36 shows the serum levels of the indicated cytokines in the indicated subjects treated with 8 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates.

Figure 37 shows the serum levels of the indicated cytokines in the indicated subjects treated with 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates.

Figures 38A - 38D show eosinophil counts in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates, As measured by cytometry or CBC (Complete Blood Count).

Figures 39A - 39D show lymphocyte counts in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates, as borrowed Measured by cytometry or CBC.

Figures 40A - 40D show peripheral CD8+ T _eff counts in indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates.

Figures 41A - 41B show Ki67 expressing CD8+ T _eff cells in indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates percentage.

Figures 42A - 42B show peripheral memory CD8+ counts in indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates.

Figures 43A - 43D show peripheral natural killer (NK) in indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates cell counts.

Figures 44A - 44B show the percentage of Ki67 expressing NK cells in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates .

Figures 45A - 45B show peripheral CD4+ T _reg counts in indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates.

Figures 46A - 46B show Ki67 expressing CD4+ T _reg cells in indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at the indicated times after administration of IL-2 conjugates percentage.

Claims (46)

A method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate, and (b) cimiprizumab, wherein: the IL -2 The conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is replaced by a structure of formula (I):

Formula (I) where: Z is _CH and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid having the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue. A method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate, and (b) cimiprizumab, wherein: the skin The cancer is unresectable skin cancer, locally advanced squamous cell carcinoma of the skin, or metastatic skin cancer; and the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is replaced by The structural substitution of formula (I):

Formula (I) where: Z is _CH and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid having the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue. A method of treating skin cancer in a subject in need thereof, comprising: selecting a subject with skin cancer, wherein the subject is selected based on one or more attributes, the attributes comprising (i) The skin cancer is unresectable skin cancer; (ii) the skin cancer is locally advanced cutaneous squamous cell carcinoma; or (iii) the skin cancer is metastatic skin cancer; and administering to the subject (a ) an IL-2 conjugate, and (b) simiprizumab, wherein: the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is represented by the formula ( I) Structural substitution:

Formula (I) where: Z is _CH and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid with the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue. A method of treating skin cancer in a subject in need thereof, comprising administering to said subject (a) about 8 μg/kg, 16 μg/kg, or 24 μg/kg of an IL-2 conjugate, and (b) simiprizumab, wherein: the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is replaced by a structure of formula (I):

Formula (I) where: Z is _CH and Y is

; Y is _CH2 and Z is

; Z is _CH2 and Y is

; or Y is _CH2 and Z is

; W is a PEG group with an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid with the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue. The method of any one of claims 1-4, wherein the cancer is melanoma. The method of any one of claims 1-4, wherein the skin cancer is squamous cell carcinoma of the skin. The method of any one of claims 1-4, wherein the skin cancer is locally advanced squamous cell carcinoma of the skin. The method of any one of claims 1-7, wherein the skin cancer is unresectable. The method of any one of claims 1-8, wherein the skin cancer is metastatic. The method of any one of claims 1-9, wherein the skin cancer is not amenable to topical therapy. The method of any one of claims 1-10, wherein the skin cancer is advanced. The method of any one of claims 1-11, wherein the skin cancer is immune checkpoint inhibitor naïve. The method of any one of claims 1-12, comprising administering to the subject about 8 μg/kg of the IL-2 conjugate. The method of any one of claims 1-12, comprising administering to the subject about 16 μg/kg of the IL-2 conjugate. The method of any one of claims 1-12, comprising administering to the subject about 24 μg/kg of the IL-2 conjugate. The method of any one of claims 1-15, wherein in the IL-2 conjugate, the PEG group has an average molecular weight of about 30 kDa. The method of any one of claims 1-16, wherein in the IL-2 conjugate, Z is CH ₂ and Y is

. The method according to any one of claims 1-16, wherein in said IL-2 conjugate, Y is CH ₂ and Z is

. The method of any one of claims 1-16, wherein in the IL-2 conjugate, Z is CH ₂ and Y is

. The method according to any one of claims 1-16, wherein in said IL-2 conjugate, Y is CH ₂ and Z is

. The method according to any one of claims 1-16, wherein the structure of formula (I) has the structure of formula (IV) or formula (V), or is a mixture of formula (IV) and formula (V):

Formula (IV);

Formula (V); wherein: W is a PEG group having an average molecular weight of about 25 kDa-35 kDa; q is 1, 2 or 3; X is an L-amino acid having the following structure:

; X-1 represents the point of attachment to the preceding amino acid residue; and X+1 represents the point of attachment to the subsequent amino acid residue. The method as described in any one of claims 1-16, wherein the structure of formula (I) has the structure of formula (XII) or formula (XIII), or is a mixture of formula (XII) and formula (XIII):

Formula (XII);

Formula (XIII); wherein: n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 30 kDa; q is 1, 2 or 3; The covalent bond of the amino acid residue being substituted. The method of any one of claims 1-22, wherein q is 1. The method of any one of claims 1-22, wherein q is 2. The method of any one of claims 1-22, wherein q is 3. The method of any one of claims 1-25, wherein the IL-2 conjugate is administered to the subject about once every two weeks, about once every three weeks, or about once every four weeks. The method of any one of claims 1-26, wherein the IL-2 conjugate and Western medicine are administered to the subject about once every two weeks, about once every three weeks, or about once every four weeks. mipilimumab. The method of any one of claims 1-27, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate or hydrate. The method of any one of claims 1-28, wherein cimiprizumab is administered every 3 weeks at a dose of about 350 mg. The method of any one of claims 1-29, wherein the IL-2 conjugate and cimiprizumab are administered separately. The method of claim 30, wherein the IL-2 conjugate and cimiprizumab are administered sequentially. The method of claim 30 or 31, wherein the IL-2 conjugate is administered prior to cimiprizumab. The method of claim 30 or 31 , wherein the IL-2 conjugate is administered after cimiprizumab. The method of any one of claims 1-33, wherein the IL-2 conjugate is administered to the subject by intravenous administration. The method of any one of claims 1-34, wherein the IL-2 conjugate and cimiprizumab are administered to the subject by intravenous administration. The method of any one of claims 1-35, further comprising administering acetaminophen to the subject. The method of any one of claims 1-36, further comprising administering diphenhydramine to the subject. The method of any one of claims 1-37, further comprising administering to the subject ondansetron. The method of any one of claims 36-38, wherein acetaminophen, diphenhydramine, and/or ondansetron are administered to the subject prior to administration of the IL-2 conjugate. The method of any one of claims 1-39, further comprising selecting to administer the IL-2 conjugate and cimiprizumab based at least in part on the skin cancer being unresectable skin cancer subject. The method of any one of claims 1-40, further comprising selectively administering the IL-2 conjugate and cimipril alone based at least in part on the skin cancer being locally advanced squamous cell carcinoma of the skin. resistant subjects. The method of any one of claims 1-41, further comprising selecting a subject for administration of the IL-2 conjugate and cimiprizumab based at least in part on the skin cancer being metastatic skin cancer tester. The method of any one of claims 1-42, further comprising selecting a subject for administration of the IL-2 conjugate and cimiprizumab based at least in part on the skin cancer being unsuitable for topical therapy By. The method of any one of claims 1-43, further comprising selectively administering the IL-2 conjugate and simetrop based at least in part on the skin cancer being immune checkpoint inhibitor naïve Limumab subjects. 1. An IL-2 conjugate for use in the method of any one of claims 1-44. Use of an IL-2 conjugate in the manufacture of a medicament for use in the method of any one of claims 1-45.

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