IMMUNOCYTOKINE CONTAINING IL-21R MUTEIN

Information

  • Patent Application
  • 20230136331
  • Publication Number
    20230136331
  • Date Filed
    September 30, 2022
    2 years ago
  • Date Published
    May 04, 2023
    a year ago
Abstract
Provided herein is an immunocytokine comprising (i) an antigen binding protein (ABP) specific to a target protein; (ii) an IL-21 domain; and (iii) an IL-21Rα mutein, wherein the IL-21Rα mutein has a reduced binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. Further provided includes a method of using the immunocytokine to selectively activate an IL-21Rα on a target cell, thereby enhance immune response or treat cancer.
Description
2. SEQUENCE LISTING

The instant application contains a sequence listing with 240 sequences which has been submitted via USPTO Patent Center is hereby incorporated by reference in its entirety. Said XML copy, created Sep. 29, 2022, is named “50042US_015US_CRF_sequencelisting.xml” and is 301,157 bytes in size.


3. BACKGROUND OF THE INVENTION

For the last decade, immune checkpoint blockade (ICB) represented by anti-PD-1 or anti-CTLA-4 antibody has led to considerable success in cancer immunotherapy, in which ICBs reprogram the immune system of patients to be against cancer. Despite the outstanding effectiveness of these types of therapeutics, few patients have benefitted from ICBs because the most patients failed to develop durable immune responses and stop the progression of cancer growth. The long-lasting and durable effector function of activated T cells is essential for eliminating cancer cells from our body through T cell-mediated immune response. In chronic infection and cancer, most of the T cells exposed to persistent antigens followed by continuous T cell receptor stimulation are exhausted. The exhausted T cells in the tumor microenvironment show dysfunction of cytokine releases like IFN-γ and TNF-α, which is their major effector function and loss of proliferation capacity. Exhausted T cells are distinguished from effector and memory T cells by high level expression of co-inhibitory receptors such as PD-1, TIM-3, or CTLA-4 on their surface. Another noticeable feature of fully differentiated exhausted T cells is epigenetic stability which might be the main reason for the resistance to ICB treatment.


A recent study done by Kristen E. Pauken reported that the epigenetic fate inflexibility of the genome of exhausted T cells impedes the transition of exhausted T cell into memory T cell, which is expected to be triggered by ICB treatment. This suggests that epigenetic reprogramming of exhausted T cells into memory T cells which have the potential for self-renewal and durable effector function, might be a solution for the limitation of current cancer immunotherapeutic.


Epigenetic reprogramming is accompanied by changes in the expression level of writer enzymes such as histone methyl transferases (HMT), histone acetyl transferases (HAT), or DNA methyltransferase (DNMT), all of which can alter the chromatin states determining the expression or suppression of a gene. It is well known that the signal triggered by a cytokine in immune cells regulates the expression level or activity of writer enzymes, which determines the differentiation fate of immune cells. From all types of cytokines, gamma chain cytokines, namely IL-2, IL-4, IL-7, IL-9, and IL-21, are known that have prominent roles in the activation of effector T cells or differentiation of memory T cells, suggesting that they can be potential candidates for anti-cancer immunotherapeutic. These cytokines can cause changes in chromosome accessibility and chromatin structure by altering the expression level of several transcription factors responsible for epigenetic modification. For example, TCF-1, a transcription factor expressed in T cells, is known that has intrinsic HDAC (histone deacetylase) activity and regulates gene expression by modifying chromatin accessibility. It was reported that the expression of TCF-1 in T cells can be induced by the treatment of cytokines like IL-7, IL-15, or IL-21 in vitro culture or in vivo experiment. Recently, lineage tracing based on single-cell sequencing analysis elucidated that TCF-1 is a key biomarker for progenitor exhausted CD4+ or CD8+ T cells (TPEX) respond to ICB treatment. This means that manipulating the expression of transcription factors like TCF-1 induced by cytokine in T cells can be another option for cancer immunotherapy. For several decades, there have been attempts to use these cytokines for cancer immunotherapy.


However, the clinical utility is minimal because of severe dose-limiting toxicities, leading a patient to death. In general, the expression of a cytokine receptor is ubiquitous all over the body, and the treatment of high doses of cytokine is related to systemic toxicities. Therefore, enhancing the specificity of a cytokine to increase the tolerable dose for systemic administration is required to solve toxicity-relating problems.


4. SUMMARY OF THE INVENTION

The present disclosure provides a novel immunocytokine specific to a target cell. The immunocytokine has activity specific to target cells by comprising a cytokine molecule (IL-21) fused to antigen binding protein (ABP) specific to a target protein and a capping moiety, interfering nonspecific binding of the cytokine molecule to a non-target cell. As a capping moiety, the present disclosure provides IL-21Rα mutein that has a reduced binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.


This immunocytokine binds to a target protein expressed on the surface of a certain cell type (e.g., immune cells) through its ABP, which results in accumulation of a cytokine close to the target cell. If a cytokine of the immunocytokine randomly binds to non-target cells before reaching to its target cell, a high dose of the cytokine might induce various side effects, and it may cause a narrow therapeutic index of the immunocytokine. To avoid this problem, the extracellular domain of IL-21Rα is used as a capping moiety to interfere with the binding of IL-21 to endogenous IL-21Rα (e.g., wild type IL21Rα (IL21RαWT)) on non-target cells. Since non-target cells lack a target protein that the ABP can recognize, the immunocytokine is not targeted to non-target cells and IL-21 stays capped by the capping moiety. Once immunocytokine with the capped IL-21 is delivered to a target cell, the capping moiety, the extracellular domain of IL-21Rα, is stripped off by competition with the endogenous IL21Rα (e.g., IL21RαWT) of a target cell, which can make IL-21 bind to the endogenous IL21Rα and transduce a signal to the target cell.


Since high binding affinity of IL-21 (approximately KD=50 pmol) to the extracellular domain of IL-21Rα can interfere with the competition between the extracellular domain of IL-21Rα of the immunocytokine and endogenous IL-21Rα of target cells, an extracellular domain of IL-21Rα in the immunocytokine was mutated (IL-21RαMutein) to have a lower binding affinity to IL-21. ABP of the immunocytokine can guide the complex comprising IL-21 and IL21RαMutein to specific target cells and the IL-21 brought to the target cells can bind and transduce signal to the target cells by competition between IL-21Rα mutein of the immunocytokine and endogenous IL-21 receptors on the surface of target cells.


Accordingly, the present disclosure provides: an immunocytokine, comprising:

    • a. an antigen binding protein (ABP) specific to a target protein;
    • b. an IL-21 domain; and
    • c. an IL-21Rα mutein,


      wherein the IL-21Rα mutein has a reduced binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.


In some embodiments, the target protein is an immune checkpoint molecule. In some embodiments, the target protein is PD-1, PD-L1, TIGIT, LAG-3, CTLA-4, TIM-3, CD39, CD38, CD73, CD36, CD25, CD47, CD24, CD20, SIPRα, CD40, or CD20.


In some embodiments, the ABP is an antibody against the target protein. In some embodiments, the ABP is an immune check point inhibitor. In some embodiments, the ABP is anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is IgG.


In some embodiments, the ABP comprises Fc fragment selected from a human IgG1 Fc fragment, a human IgG2 Fc fragment, a human IgG3 Fc fragment, and a human IgG4 Fc fragment. In some embodiments, the Fc fragment is a human IgG4 Fc fragment. In some embodiments, the Fc fragment comprises the sequence selected from SEQ ID NOs: 16, 185-190.


In some embodiments, the ABP comprises an Fc fragment with two Fc moieties. In some embodiments, the IL-21Rα mutein is linked to the first of the two Fc moieties, and the IL-21 domain is linked to the second of the two Fc moieties. In some embodiments, the IL-21 domain and the IL-21Rα mutein are respectively linked through a non-cleavable peptide linker or without a peptide linker. In some embodiments, the non-cleavable peptide linker is G4S linker having the sequence of SEQ ID NO: 17. In some embodiments, the non-cleavable peptide linker has a sequence selected from SEQ ID NOs: 212-224.


In some embodiments, the ABP is selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tremelimumab, tiragolumab, relatlimab, or a functional variant thereof. In some embodiments, the ABP comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences of nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, or tremelimumab. In some embodiments, the ABP comprises heavy chain and/or light chain of nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, or tremelimumab. In some embodiments, the ABP comprises a heavy chain variable domain and/or a light chain variable domain of nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, or tremelimumab. In some embodiments, the heavy chain variable domain and/or the light chain domain are linked to a human IgG1 Fc fragment, a human IgG2 Fc fragment, a human IgG3 Fc fragment, or a human IgG4 Fc fragment. In some embodiments, the Fc fragment includes a mutation for knob-in-hole interaction,


In some embodiments, the ABP comprises:

    • a. a heavy chain having the sequence of SEQ ID NO: 1 and a light chain having the sequence of SEQ ID NO: 2;
    • b. a heavy chain having the sequence of SEQ ID NO: 3 and a light chain having the sequence of SEQ ID NO: 4;
    • c. a heavy chain having the sequence of SEQ ID NO: 5 and a light chain having the sequence of SEQ ID NO: 6;
    • d. a heavy chain having the sequence of SEQ ID NO: 7 and a light chain having the sequence of SEQ ID NO: 8;
    • e. a heavy chain having the sequence of SEQ ID NO: 9 and a light chain having the sequence of SEQ ID NO: 10;
    • f. a heavy chain having the sequence of SEQ ID NO: 11 and a light chain having the sequence of SEQ ID NO: 12;
    • g. a heavy chain having the sequence of SEQ ID NO: 13 and a light chain having the sequence of SEQ ID NO: 14;
    • h. a heavy chain having the sequence of SEQ ID NO: 151 and a light chain having the sequence of SEQ ID NO: 152;
    • i. a heavy chain having the sequence of SEQ ID NO: 153 and a light chain having the sequence of SEQ ID NO: 154;
    • j. a heavy chain having the sequence of SEQ ID NO: 225 and a light chain having the sequence of SEQ ID NO: 226; or
    • k. a heavy chain having the sequence of SEQ ID NO: 227 and a light chain having the sequence of SEQ ID NO: 228.


In some embodiments, the ABP comprises:

    • a. a heavy chain having the sequence of SEQ ID NO: 1 or a variation thereof, and a light chain having the sequence of SEQ ID NO: 2, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 1;
    • b. a heavy chain having the sequence of SEQ ID NO: 3 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 4, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 3;
    • c. a heavy chain having the sequence of SEQ ID NO: 5 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 6, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 5;
    • d. a heavy chain having the sequence of SEQ ID NO: 7 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 8, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 7;
    • e. a heavy chain having the sequence of SEQ ID NO: 9 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 10, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 9;
    • f. a heavy chain having the sequence of SEQ ID NO: 11 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 12, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 11;
    • g. a heavy chain having the sequence of SEQ ID NO: 13 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 14, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 13;
    • h. a heavy chain having the sequence of SEQ ID NO: 151 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 152, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 151;
    • i. a heavy chain having the sequence of SEQ ID NO: 153 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 154, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 153;
    • j. a heavy chain having the sequence of SEQ ID NO: 225 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 226, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID 225; or
    • k. a heavy chain having the sequence of SEQ ID NO: 227 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 228, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID 227.


In some embodiments, the IL-21Rα mutein has at least 10-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 10,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 500 to 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.


In some embodiments, the IL-21Rα mutein has a sequence with at least 95% sequence identity to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein has a sequence with at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15 (IL-21Rα WT).


In some embodiments, the IL-21Rα mutein comprises at least one amino acid substitution compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein comprises one to five amino acid substitutions compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein comprises one amino acid substitution compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94 of the wild-type IL-21Rα sequence.


In some embodiments, the amino acid substitutions are selected from:

    • a. Y10A
    • b. Q35K, Q35R, or Q35Y;
    • c. Y36A, Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;
    • d. E38A, E38C, E38K, E38R, or E38Y;
    • e. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;
    • f. F67A;
    • g. H68A;
    • h. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;
    • i. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;
    • j. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;
    • k. D73C, D73A, D73E, D73H, D73K, D73R, D73W, or D73Y;
    • l. I74A, I74H, I74K, I74R, or I74W;
    • m. L94A, L94F, L94K, L94Q, L94R, or L94Y;
    • n. P126A;
    • o. Y129A;
    • p. M130A;
    • q. K134A;
    • r. 5189A;
    • s. 5190A; and
    • t. Y191A.


In some embodiments, the amino acid substitutions are selected from:

    • a. Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;
    • b. E38C, E38R, or E38Y;
    • c. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;
    • d. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;
    • e. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;
    • f. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;
    • g. D73A
    • h. I74R, or I74W; and
    • i. L94A, L94F, L94K, L94Q, L94R, or L94Y.


In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169.


In some embodiments, the immunocytokine comprises a first chain comprising from the N terminus to C terminus:

    • a. a Fc fragment of a human IgG1, IgG2, IgG3 or IgG4 having any one sequence selected from SEQ ID NOs: 16, and 185-190; and
    • b. an IL-21Rα mutein having a sequence selected from SEQ ID NOs: 18-99 and 155-169.


In some embodiments, the immunocytokine comprises a first chain comprising from the N terminus to C terminus:

    • a. a Fc fragment of a human IgG1, IgG2, IgG3 or IgG4 having any one sequence selected from SEQ ID NOs: 16, and 185-190;
    • b. a peptide linker; and
    • c. an IL-21Rα mutein having a sequence selected from SEQ ID NOs: 18-99 and 155-169.


In some embodiments, the immunocytokine comprises a first chain comprising from the N terminus to C terminus:

    • a. a heavy chain of the ABP comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225, 227, or a variant thereof; and
    • b. an IL-21Rα mutein.


In some embodiments, the immunocytokine comprises a first chain comprising from the N terminus to C terminus:

    • a. a heavy chain of the ABP comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225, 227, or a variant thereof;
    • b. a peptide linker; and
    • c. an IL-21Rα mutein.


In some embodiments, the heavy chain of the ABP comprises a knob variant or a hole variant for knobs-in-holes interaction, wherein the knob variant and the hole variant comprise one or more modifications for the knobs-in-holes interaction.


In some embodiments, the heavy chain of the ABP comprises a variant of the sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227, wherein the variant has deletion of Lys (K) at the C-terminal end of the sequence.


In some embodiments, the heavy chain of the ABP comprises the sequence of SEQ ID NO: 103.


In some embodiments, the peptide linker is a G45 linker having the sequence of SEQ ID NO: 17. In some embodiments, the peptide linker has a sequence selected from SEQ ID NOs: 212-224.


In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169. In some embodiments, the first chain has a sequence selected from SEQ ID NOs: 104-150 and 192-209.


In some embodiments, the immunocytokine comprises a second chain comprising a heavy chain of the ABP, a peptide linker and the IL-21 domain. In some embodiments, the heavy chain of the ABP comprising a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. In some embodiments, the heavy chain of the ABP comprises a variant of the sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. The variant comprises deletion of lysine (Lys or K) at the C-terminal end of the sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. In some embodiments, the heavy chain of the ABP may comprise a knob variant or a hole variant for knobs-in-holes interaction. In some embodiments, the peptide linker is selected from SEQ ID NO: 17 and SEQ ID NOs: 212-224. In some embodiments, the second chain has the sequence of SEQ ID NO: 101. In some embodiments, the IL-21 domain is a human IL-21 or a functional variant thereof. In some embodiments, the IL-21 domain has the sequence of SEQ ID NO: 100 (human IL-21).


In some embodiments, the immunocytokine comprises a first heavy chain and a second heavy chain of the ABP. In some embodiments, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation for knob-and-hole interaction. In some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation for knob-and-hole interaction. In some embodiments, the heavy chain is full length heavy chain or the fragment thereof. In some embodiments, the hole mutation and knob mutation are comprised in a Fc moiety of each heavy chain. In some embodiments, the hole mutation and knob mutation are comprised in a CH3 domain of each heavy chain.


In some embodiments, the immunocytokine comprises a light chain having the sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 102, 152, 154, 226 and 228.


In some embodiments, the IL-21 domain is a human IL-21 or a functional variant thereof. In some embodiments, the IL-21 domain has the sequence of SEQ ID NO: 100 (human IL-21).


In another aspect, the present disclosure provides one or more polynucleotides encoding the immunocytokine provided herein.


In some embodiments, the one or more polynucleotides comprise:

    • a. a first polynucleotide segment encoding a first chain comprising the heavy chain of the ABP and the IL-21Rα mutein;
    • b. a second polynucleotide segment encoding a second chain comprising the heavy chain of the ABP and the IL-21 domain; and
    • c. a third polynucleotide segment encoding the light chain of the ABP.


In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are in a single polynucleotide molecule. In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are in multiple polynucleotide molecules. In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are individually present in separate polynucleotide molecules.


In another aspect, the present disclosure provides one or more vectors comprising the one or more polynucleotides described herein.


In yet another aspect, the present disclosure provides a host cell comprising the one or more polynucleotides or the one or more vectors described herein. In some embodiments, host cell comprises the immunocytokine provided herein. In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a T cell.


The host cell can be a eukaryotic cell, for example a fungal cell such as yeast. The host cell can be a mammalian cell (which may be a cell in cell culture, or a cell present in a tissue or organ). In some embodiments, the host cell is a human, mouse, rat, rabbit, bovine or dog (or, for example, any other wild, livestock/domesticated animal) cell. In some embodiments, the host cell is a stable cell line cell, or a primary cell, adherent or suspension cell. As examples, the host cell can be a macrophage, osteosarcoma, or CHO, BHK (baby hamster kidney), Bowes human melanoma cell, 911, AT1080, A549, HEK293, or HeLa cell line cell or a mouse primary cell, but not limited thereto. In some embodiments, the host cell is a bacterial cell, such as E. coli.


The eukaryotic cell can be a plant cell (for example a monocotyledonous or dicotyledonous plant cell; typically an experimental, crop and/or ornamental plant cell, for example Arabidopsis, maize); fish (for example Zebra fish; salmon), bird (for example chicken or other domesticated bird), insect (for example Drosophila; bees), Nematoidia or Protista (for example Plasmodium spp or Acantamoeba spp) cell.


In one aspect, the present disclosure provides a method of enhancing immune response in a subject, comprising administration of the immunocytokine described herein or the host cell described herein to the subject. In some embodiments, the subject is a cancer patient.


In one aspect, the present disclosure provides a method of selectively activating an IL-21Rα on a target cell, comprising: delivering the immunocytokine of the present disclosure to the target cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell.


Another aspect of the present disclosure provides an IL-21Rα mutein having a reduced binding affinity to an IL-21 domain compared to a wild-type IL-21Rα.


In some embodiments, the wild-type IL-21Rα comprises the sequence of SEQ ID NO: 15. In some embodiments, the IL-21 domain is a human IL-21 or a functional variant thereof. In some embodiments, the IL-21 domain has the sequence of SEQ ID NO: 100.


In some embodiments, the IL-21Rα mutein has at least 10-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 10,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 500 to 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.


In some embodiments, the IL-21Rα mutein has about 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.


In some embodiments, the IL-21Rα mutein has a sequence with at least 95% sequence identity to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein has a sequence with at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15 (IL-21Rα WT).


In some embodiments, the IL-21Rα mutein comprises at least one amino acid substitution compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein comprises one to five amino acid substitutions compared to SEQ ID NO: 15 (IL-21Rα WT). In some embodiments, the IL-21Rα mutein has one amino acid substitution compared to SEQ ID NO: 15 (IL-21Rα WT).


In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94 of the wild-type IL-21Rα sequence.


In some embodiments, the amino acid substitutions are selected from:

    • a. Y10A
    • b. Q35K, Q35R, or Q35Y;
    • c. Y36A, Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;
    • d. E38A, E38C, E38K, E38R, or E38Y;
    • e. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;
    • f. F67A;
    • g. H68A;
    • h. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;
    • i. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;
    • j. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;
    • k. D73C, D73A, D73E, D73H, D73K, D73R, D73W, or D73Y;
    • l. I74A, I74H, I74K, I74R, or I74W;
    • m. L94A, L94F, L94K, L94Q, L94R, or L94Y;
    • n. P126A;
    • o. Y129A;
    • p. M130A;
    • q. K134A;
    • r. S189A;
    • s. S190A; and
    • t. Y191A.


In some embodiments, the amino acid substitutions are selected from:

    • a. Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;
    • b. E38C, E38R, or E38Y;
    • c. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;
    • d. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;
    • e. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;
    • f. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;
    • g. D73A
    • h. I74R, or I74W; and
    • i. L94A, L94F, L94K, L94Q, L94R, or L94Y.


In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169.


In another aspect, the present disclosure provides a polynucleotide comprising a coding sequence of the IL-21 Rα mutein described herein. In yet another aspect, the present disclosure provides a vector comprising the polynucleotide. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a recombinant AAV or lentiviral vector.


The present disclosure also provides a host cell comprising the IL-21 Rα mutein, the polynucleotide, or the vector described herein.





5. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:



FIG. 1 provides a schematic representation of an exemplary immunocytokine (αPD-1IL21RαMutein/IL21).



FIGS. 2A-2V provide sensorgrams from SPR full kinetics assay of IL-21Rα muteins against IL21.



FIG. 3 provides experimental results testing 66 different αPD-1IL21RαMutein/IL21. X-axis shows the affinity of immunocytokines to IL21 measured by Bio Layer interferometry (BLI) and y-axis shows efficacy of IL-21 mediated STAT3 phosphorylation (efficacy coefficient). The results show that as IL21Rα mutein in the immunocytokine has a reduced binding affinity to IL-21, the immunocytokine (αPD-1IL21RαMutein/IL21) has a higher efficacy coefficient.



FIG. 4 provides concentration dependent curves of selected six αPD-1IL21RαMutein/IL21, αPD-1IL21RαWT/IL21 (“WT”) and recombinant human IL-21 protein (“IL-21”). Specifically, the graph shows IL-21 mediated activation (efficacy coefficient) in PD-1(+) H9 cells. The max potency of αPD-1IL21RαMutein/IL21 with M70Q and M70H mutation was comparable to the recombinant human IL-21 (“IL-21”), and the others showed at least above 80%.



FIG. 5 provides concentration dependent curves of selected six αPD-1IL21RαMutein/IL21, αPD-1IL21RαWT/IL21 (“WT”) and recombinant human IL-21 protein (“IL-21”). Specifically, the graph shows IL-21 mediated activation (efficacy coefficient) in PD-1(−) H9 cells. The max potency of six αPD-1IL21RαMutein/IL21 was similar to the recombinant human IL-21 (“IL-21”), but EC50 increased in all variants.



FIGS. 6A-6E provide response curve of STAT3 phosphorylation observed in PD-1(−) H9 cells and PD-1(+) H9 cells in response to 16 variants of αPD-1IL21RαMutein/IL21.



FIGS. 7A-7Q provide sensorgrams from SPR full kinetics assay of immunocytokines (αPD-1IL21RαMutein/IL21) against PD-1.



FIG. 8A shows measurements of the binding affinities of the αPD-1 antibody or Immunocytokine to FcRn using ForteBio Octet RED96e instruments. FIG. 8B is a table summarizing binding kinetics of the αPD-1 antibody or Immunocytokine to FcRn.



FIGS. 9A, 9B and 9C provide sensorgrams data from SPR full kinetics assay of immunocytokines (αCTLA-4IL21RαMutein/IL21; αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21) against their targets (hCTLA-4, hTIGIT, or hLAG-3).



FIG. 10 shows IFNγ concentrations (pg/ml) released from CTLs (effector cells) co-cultured with immunocytokines (αPD-1IL21RαMutein/IL21 including M70D, M70Q, L94K and E38R) as described in Section 5.7. The data are compared against IFNγ release in response to αPD-1 antibody or αPD-1IL21RαWT/IL21 (“WT”).



FIGS. 11A and 11B show fluorescent signals (RFU) of Calcein AM released from dead tumor cells as described in Section 5.7.2. The signal indicates tumor killing efficacy of effector cells against tumor cells (MeWo cell line (FIG. 11A) and A375_CMV cell line (FIG. 11B)) treated with immunocytokines (αPD-1IL21RαMutein/IL21 including M70D, M70Q, L94K and E38R) or controls (αPD-1 antibody or αPD-1IL21RαWT/IL21 (“WT”)).



FIG. 12 provides STAT3 phosphorylation curves obtained from HTRF-based high-throughput assay described in Section 5.9. It shows STAT3 phosphorylation induced by rhIL21, ABP-IL21RαWT/IL21, and ABP-IL21RαMutein/IL21, but not by antibodies without IL21 conjugation (i.e., anti-CTLA-4 antibody (Ipilimumab), anti-TIGIT antibody (Tiragolumab), anti-LAG-3 antibody (Relatlimab)).





6. DETAILED DESCRIPTION OF THE INVENTION

6.1. Definitions


The term “IL-21Rα mutein”, “IL-21RαMutein”, “IL21Rα mutein” or “IL21RαMutein” as used herein refers to the ectodomain of an IL-21Rα having one or more modifications. The modifications can be amino acid substitution, insertion, deletion or other mutation. In some embodiments, IL-21Rα mutein includes one or more biological, chemical, or both modifications compared to wild-type human IL-21Rα or its ectodomain. In some embodiments, the ectodomain of the wild-type human IL-21Rα comprises the sequence of SEQ ID NO: 15.


The term “pharmaceutically acceptable carrier” as used herein refers to a carrier or diluent that does not impair the biological activity and characteristics of an immunocytokine according to the present invention. As a pharmaceutically acceptable carrier in a composition that is formulated as a liquid solution, a sterile and biocompatible carrier can be used. The pharmaceutically acceptable carrier can be physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of two or more thereof. In addition, the composition of the present invention may, if necessary, comprise other conventional additives, including antioxidants, buffers, and bacteriostatic agents. Further, the composition of the present invention can be formulated as injectable forms such as aqueous solutions, suspensions or emulsions with the aid of diluents, dispersants, surfactants, binders and lubricants. In addition, the composition according to the present invention can be formulated in the form of pills, capsules, granules, or tablets. Other carriers known in the art, e.g., as described in a literature [Remington's Pharmaceutical Sciences (E. W. Martin)], can be used.


The term “antigen-binding protein (ABP)” refers to a protein comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP consists of an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. In some embodiments, the ABP binds the extracellular domain of the target protein. In certain embodiments, the ABP provided herein binds to an epitope of the target protein that is conserved between or among various species.


In some embodiments, the ABP is an antibody and the antibody can be a monoclonal antibody, a polyclonal antibody, a multi-specific antibody, a dual-specific or bispecific antibody, an anti-idiotypic antibody, or a bifunctional hybrid antibody. In some embodiments, the ABP comprises one or more heavy chain or a fragment thereof. In some embodiments, the ABP comprises one or more light chain or a fragment thereof. In some embodiments, the antibody comprises two heavy chains and two light chains, or fragments thereof. In some embodiments, the fragment of the heavy chain comprises Fc fragment, CH3 domain, or CH2 domain of the heavy chain.


The term “alternative scaffold” refers to a molecule in which one or more regions may be diversified to produce one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins™), the β-sandwich (e.g., iMab), lipocalin (e.g., Anticalins®), EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domains), thioredoxin peptide aptamers, protein A (e.g., Affibody®), ankyrin repeats (e.g., DARPins), diabody, gamma-B-crystallin/ubiquitin (e.g., Affilins), CTLD3 (e.g., Tetranectins), Fynomers, and LDLR-A module (e.g., Avimers). Additional information on alternative scaffolds is provided in Binz et al., Nat. Biotechnol., 2005 23:1257-1268; Skerra, Current Opin. In Biotech., 2007 18:295-304; and Silacci et al., J. Biol. Chem., 2014, 289:14392-14398; each of which is incorporated by reference in its entirety. An alternative scaffold is one type of ABP.


The term “antibody fragment” comprises a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, antigen-binding fragments (Fab), F(ab′)2 fragments, Fab′ fragments, single chain variable fragments (scFv, sFv), scFv-Fc fragments. Disulfide-linked Fv fragments, and a single domain antibody (sdAb).


The term “antigen-binding domain” means the portion of an ABP that is capable of specifically binding to an antigen or epitope.


The term “Fe fragment” means the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system. The structures of the Fc regions of various immunoglobulins, and the glycosylation sites contained therein, are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125: S41-52, incorporated by reference in its entirety. The Fc fragment can comprise two Fc moieties. The Fc moiety can comprise a CH2-CH3 domain of a heavy chain. In some embodiments, the ABP comprises an Fc fragment comprising two Fc moieties, wherein each Fc moiety is independently selected from IgG subclasses, e.g., IgG1, IgG2, IgG3, and IgG4. In some embodiments, the ABP comprises two Fc moieties of IgG1. In some embodiments, the ABP comprises two Fc moieties of IgG4. In some embodiments, the ABP comprises an Fc fragment comprising two Fc moieties, wherein the first Fc moiety is an Fc moiety of IgG1 and the second Fc moiety is an Fc moiety of IgG4. In some embodiments, the ABP comprises an Fc fragment comprising two Fc moieties, wherein the first Fc moiety comprises an CH3 of IgG1 and the second Fc moiety comprises an CH3 of IgG4.


The Fc region may be a naturally occurring Fc region, or an Fc region modified as described elsewhere in this disclosure. For example, the Fc moiety can be a knob variant or a hole variant for knobs-in-holes interaction. The Fc fragment can comprise a knob variant and a hole variant of a C-terminal region of an immunoglobulin heavy chain.


In some cases, the Fc fragment is engineered to introduce mutations to reduce effector function of immunoglobulin, which minimize ADCC by reducing the binding affinity for FcγR. Those mutations are the so-called LALA mutation(L234A/L235A) for human IgG1 type and SPLE mutation (S228P/L235E). (see, e.g., Hezareh et al. J. Virol. (2001) 75(24): 12161-8). In further embodiments, the LALA or SPLE mutations are present in the Fc fragment with the knobs-into-holes mutations.


The Fc fragment can comprise the M252Y/S254T/T256E (“YTE”) mutations. The YTE mutations allow the simultaneous modulation of serum half-life, tissue distribution and activity of IgG1 (see DalFAcqua et al., J Biol Chem. (2006) 281:23514-24; and Robbie et al., Antimicr oh Agents Chemother. (2013) 57(12):6147-53). In further embodiments, the YTE mutations are present in the antibody with the knobs-into-holes mutations.


The VH and VL regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding, and influence antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, Md., incorporated by reference in its entirety.


The light chain from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of its constant domain.


The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.


The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Plückthun, J. Mol. Biol., 2001, 309:657-70 (“Aho” numbering scheme); each of which is incorporated by reference in its entirety.


Table 1 provides exemplary positions of CDR1-L (CDR1 of VL), CDR2-L (CDR2 of VL), CDR3-L (CDR3 of VL), CDR1-H (CDR1 of VH), CDR2-H (CDR2 of VH), and CDR3-H (CDR3 of VH), as identified by the Kabat and Chothia schemes. For CDR1-H, residue numbering is provided using both the Kabat and Chothia numbering schemes.


CDRs may be assigned, for example, using antibody numbering software, such as Abnum, available at www.bioinf.org.uk/abs/abnum/, and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839 or bioinf.org.uk—Prof. Andrew C. R. Martin's group at UCL, incorporated by reference in its entirety.









TABLE 1







Exemplary CDR residues according to


Kabat and Chothia numbering schemes.











CDR
Kabat
Chothia







CDR1-L
24-34
24-34



CDR2-L
50-56
50-56



CDR3-L
89-97
89-97



CDR1-H (Kabat Numbering)
31-35B
26-32 or 34*



CDR1-H (Chothia Numbering)
31-35
26-32



CDR2-H
50-65
52-56



CDR3-H
 95-102
 95-102







*The C-terminus of CDR1-H, when numbered using the Kabat numbering convention, varies between 32 and 34, depending on the length of the CDR.






The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminish of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.


6.2. Other Interpretational Conventions


Ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.


Unless otherwise indicated, reference to a compound that has one or more stereocenters intends each stereoisomer, and all combinations of stereoisomers, thereof.


6.3. Summary of Experimental Observation


The present disclosure provides IL21RαMutein having a reduced affinity to IL-21 compared to IL21RαWT, and immunocytokines comprising the IL21RαMutein as a capping moiety.


As one example, the present disclosure provides an immunocytokine (αPD-1IL21RαMutein/IL21) comprising an ABP targeting PD-1. The immunocytokine can be targeted to PD-1 expressing cells, such as CD4+ or CD8 T+ cells. The immunocytokine comprises four polypeptide chains—two identical light chains and two different heavy chains—joined to form a heterodimer by knobs-into-holes (KiH) interaction. In the immunocytokine, one of the two heavy chains is fused to IL-21 and the other one is fused to a capping moiety, which is a mutant of ectodomain of IL-21Rα (IL-21RαMutein). IL-21 and the capping moiety are fused to the heavy chains through a non-cleavable and flexible polypeptide linker.


Applicant expressed the immunocytokines in CHO cells and purified them with a purity of ≥95%. The immunocytokine had 185 kDa molecular weight in the de-glycosylated form and 195 kDa in the glycosylated form when measured by mass spectrometry. Applicant also confirmed that over 90% of the molecules were present in the heterodimeric form of αPD-1IL21RαMutein/IL21. Applicant further measured activity of anti-PD-1 antibody using the PD-L1/TCR activator-CHO recombinant cell line (BPS bioscience) which can measure the intensity of TCR signaling through a luciferase reporter system driven by an NFAT-response element. The experiment showed that the fusion of IL21RαMutein/IL21 to IgG had little effect on the activity of anti-PD-1 antibody. Furthermore, SPR analysis demonstrated that the fusion of IL-21WT or IL21RαMutein and IL-21 to the anti-PD-1 antibody did not affect the affinity to PD-1 (FIG. 7A-7Q).


Applicant generated 66 candidates of immunocytokine comprising anti-PD-1 IgG, IL-21, and one of various muteins of IL-21Rα. Next, using a high throughput HTRF assay, Applicant tested whether application of αPD-1IL21RαMutein/IL21 increases phosphorylation of STAT3 in PD-1(+) T cell. Based on the results from the HTRF assay, six αPD-1IL21RαMutein/IL21 candidates were selected. The selected candidates showed max potency at lower concentration compared to control αPD-1IL21RαWT/IL21 treatment and acted selectively on PD-1(+) cells. They showed superior potency at lower concentration compared to the control immunocytokine (αPD-1IL21RαWT/IL21).


Anti-cancer efficacy of the selected candidates can be tested in a humanized PDX mouse model. When αPD-1IL21RαMutein/IL21 binds to PD-1 expressed on PD-1(+) T cells, the reduced binding affinity of IL21RαMutein to human IL-21 can allow IL-21 of the immunocytokine to compete with and bind to endogenous IL21Rα (e.g., IL21RαWT), and lead to the invigoration of PD-1(+) T cells for the generation of durable anti-cancer immunity.


In summary, the present disclosure provides an immunocytokine that can exclusively deliver IL-21 to PD-1(+) T cells and reinvigorate the T cells to acquire a memory-like phenotype for long-lasting anti-cancer immunity.


6.4. IL-21Rα Muteins


In one aspect, the present disclosure provides IL-21Rα muteins having a reduced binding affinity to an IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has a mutation at the binding site of IL-21Rα against IL-21. In some embodiments, the IL-21Rα mutein has one or more amino acid substitution, insertion, or deletion at a binding site of IL-21Rα against IL-21.


In some embodiments, the IL-21Rα mutein specifically binds to the IL-21 domain, but with a reduced affinity. In some embodiments, the IL-21Rα mutein has at least 10-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 50-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 200-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 300-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has at least 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.


In some embodiments, the IL-21Rα mutein has 10 to10,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 5,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 5,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 10 to 1,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 100 to 1,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 500 to 1,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 500 to 2,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 1,000 to 2,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has 2,000 to 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.


In some embodiments, the IL-21Rα mutein has about 5000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 2500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 1000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 500-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα. In some embodiments, the IL-21Rα mutein has about 100-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.


In some embodiments, the wild-type IL-21Rα is the ectodomain of a human IL-21Rα. In some embodiments, the wild-type IL-21Rα has the sequence of SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 95% sequence identity to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 96% sequence identity to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 97% sequence identity to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 98% sequence identity to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein has a sequence with at least 99% sequence identity to SEQ ID NO: 15.


In some embodiments, the IL-21 Rα mutein includes one or more modifications at a binding site involved in the interaction between IL-21 and IL-21 Rα. In some embodiments, the one or more modifications are amino acid substitution, deletion, insertion, or a combination thereof. In some embodiments, the one or more modifications are chemical modifications. In some embodiments, the modifications can induce structural change in the binding site.


In some embodiments, the IL-21Rα mutein comprises at least one amino acid substitution compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises one amino acid substitution compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises two amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises three amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises four amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises five amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises more than five amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the IL-21Rα mutein comprises one to five amino acid substitutions compared to SEQ ID NO: 15.


In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the IL-21Rα mutein comprises an amino acid substitution at one amino acid position selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence.


In some embodiments, the one or more amino acid substitutions are at one or more amino acid positions selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94 of the wild-type IL-21Rα sequence. In some embodiments, the IL-21Rα mutein comprises an amino acid substitution at one amino acid position selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94 of the wild-type IL-21Rα sequence.


In some embodiments, the IL-21 Rα mutein comprises a sequence different from the wild-type IL-21Rα sequence only at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the IL-21 Rα mutein comprises a sequence different from the wild-type IL-21Rα sequence only at one or more amino acid positions selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94.


In some embodiments, the IL-21 Rα mutein comprises a sequence different from the wild-type IL-21Rα sequence only at one amino acid position selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 of the wild-type IL-21Rα sequence. In some embodiments, the IL-21 Rα mutein comprises a sequence different from the wild-type IL-21Rα sequence only at one amino acid position selected from Y36, E38, L39, M70, A71, D72, D73, I74, and L94.


In some embodiments, the one or more amino acid substitutions are selected from:

    • a. Y10A
    • b. Q35K, Q35R, or Q35Y;
    • c. Y36A, Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;
    • d. E38A, E38C, E38K, E38R, or E38Y;
    • e. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;
    • f. F67A;
    • g. H68A;
    • h. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;
    • i. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;
    • j. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;
    • k. D73C, D73A, D73E, D73H, D73K, D73R, D73W, or D73Y;
    • l. I74A, I74H, I74K, I74R, or I74W;
    • m. L94A, L94F, L94K, L94Q, L94R, or L94Y;
    • n. P126A;
    • o. Y129A;
    • p. M130A;
    • q. K134A;
    • r. S189A;
    • s. S190A; and
    • t. Y191A.


In some embodiments, the one or more amino acid substitutions are selected from:

    • a. Y36C, Y36E, Y36G, Y36H, Y36I, Y36K, Y36M, Y36N, Y36P, Y36Q, Y36R, Y36S, Y36T, or Y36V;
    • b. E38C, E38R, or E38Y;
    • c. L39A, L39C, L39E, L39F, L39H, L39K, L39R, L39W, or L39Y;
    • d. M70C, M70D, M70F, M70G, M70H, M70K, M70L, M70N, M70Q, M70R, M70S, M70T, M70V, M70W, or M70Y;
    • e. A71E, A71F, A71I, A71L, A71Q, A71R, A71W, or A71Y;
    • f. D72A, D72C, D72E, D72F, D72G, D72H, D72I, D72K, D72L, D72M, D72Q, D72R, D72W, or D72Y;
    • g. D73A
    • h. I74R, or I74W; and
    • i. L94A, L94F, L94K, L94Q, L94R, or L94Y.


In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169. In some embodiments, the IL-21Rα mutein comprises a functional fragment of a protein having a sequence selected from SEQ ID NOs: 18-99 and 155-169. The functional fragment can bind to the IL-21 domain.


6.5. Immunocytokines


In another aspect, the present disclosure provides an immunocytokine comprising: (i) an antigen binding protein (ABP) specific to a target protein; (ii) an IL-21 domain; and (iii) an IL-21Rα mutein, wherein the IL-21Rα mutein has a reduced binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.


The immunocytokine can comprise an IL-21Rα mutein disclosed in section 6.4. In some embodiments, the immunocytokine is one selected from R-kine-1 to 66.


6.5.1. Antigen Binding Protein (ABP)


The immunocytokine disclosed herein comprises an antigen binding protein (ABP) specific to a target protein.


The target protein can be a surface protein of an immune cell. In some embodiments, the target protein is a surface protein specific to a T cell. In some embodiments, the target protein is specific to CD4+ or CD8 T+ cells.


In some embodiments, the target protein is an immune checkpoint molecule. In some embodiments, the target protein is PD-1, PD-L1, TIGIT, LAG-3, CTLA-4, TIM-3, CD39, CD38, CD73, CD36, CD25, CD47, CD24, CD20, SIPRα, CD40, or CD20.


In some embodiments, the ABP is an antibody against the target protein or a fragment thereof.


In some embodiments, the ABP is an immune check point inhibitor. In some embodiments, the ABP is anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is IgG. In some embodiments, the ABP is anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is IgG. In some embodiments, the ABP is anti-TIGIT antibody. In some embodiments, the anti-TIGIT antibody is IgG. In some embodiments, the ABP is anti-LAG-3 antibody. In some embodiments, the anti-LAG-3 antibody is IgG.


In some embodiments, the ABP comprises Fc fragment selected from a human IgG1 Fc fragment, a human IgG2 Fc fragment, a human IgG3 Fc fragment, and a human IgG4 Fc fragment. In some embodiments, the Fc fragment is a human IgG4 Fc fragment. In some embodiments, the Fc fragment is a human IgG1 Fc fragment. In some embodiments, the Fc fragment comprises a modification for knob-hole interaction. In some embodiments, the Fc fragment is engineered to introduce mutations to reduce effector function of immunoglobulin, which minimize ADCC by reducing the binding affinity for FcγR. In some embodiments, the Fc fragment comprises the sequence selected from SEQ ID NOs: 16, 185-190. In some embodiments, the Fc fragment is engineered to increase stability of the Fc fragment or the immunocytokine containing the Fc fragment. For example, the Fc fragment is engineered to remove Lys (K) at the C-terminal end.


In some embodiments, the ABP is selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tremelimumab, tiragolumab, relatlimab, or a functional variant thereof. A functional variant refers to an ABP having one or more modification compared to an original ABP but maintaining the binding affinity and/or specificity of the original ABP. In some embodiments, the functional variant comprises a binding domain of the original ABP and a heterologous Fc fragment.


In some embodiments, the ABP comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences of an antibody selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, tremelimumab. In some embodiments, the ABP comprises a heavy chain variable domain of an antibody selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, tremelimumab. In some embodiments, the ABP comprises a light chain variable domain of an antibody selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, tremelimumab. In some embodiments, the ABP comprises a heavy chain variable domain and a light chain variable domain of an antibody selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, tremelimumab.


In some embodiments, the ABP comprises:

    • a. a heavy chain having the sequence of SEQ ID NO: 1 and a light chain having the sequence of SEQ ID NO: 2;
    • b. a heavy chain having the sequence of SEQ ID NO: 3 and a light chain having the sequence of SEQ ID NO: 4;
    • c. a heavy chain having the sequence of SEQ ID NO: 5 and a light chain having the sequence of SEQ ID NO: 6;
    • d. a heavy chain having the sequence of SEQ ID NO: 7 and a light chain having the sequence of SEQ ID NO: 8;
    • e. a heavy chain having the sequence of SEQ ID NO: 9 and a light chain having the sequence of SEQ ID NO: 10;
    • f. a heavy chain having the sequence of SEQ ID NO: 11 and a light chain having the sequence of SEQ ID NO: 12;
    • g. a heavy chain having the sequence of SEQ ID NO: 13 and a light chain having the sequence of SEQ ID NO: 14;
    • h. a heavy chain having the sequence of SEQ ID NO: 151 and a light chain having the sequence of SEQ ID NO: 152;
    • i. a heavy chain having the sequence of SEQ ID NO: 153 and a light chain having the sequence of SEQ ID NO: 154;
    • j. a heavy chain having the sequence of SEQ ID NO: 225 and a light chain having the sequence of SEQ ID NO: 226; or
    • k. a heavy chain having the sequence of SEQ ID NO: 227 and a light chain having the sequence of SEQ ID NO: 228.


In some embodiments, the ABP comprises:

    • a. a heavy chain having the sequence of SEQ ID NO: 1 or a variation thereof, and a light chain having the sequence of SEQ ID NO: 2, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 1;
    • b. a heavy chain having the sequence of SEQ ID NO: 3 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 4, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 3;
    • c. a heavy chain having the sequence of SEQ ID NO: 5 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 6, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 5;
    • d. a heavy chain having the sequence of SEQ ID NO: 7 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 8, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 7;
    • e. a heavy chain having the sequence of SEQ ID NO: 9 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 10, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 9;
    • f. a heavy chain having the sequence of SEQ ID NO: 11 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 12, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 11;
    • g. a heavy chain having the sequence of SEQ ID NO: 13 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 14, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 13;
    • h. a heavy chain having the sequence of SEQ ID NO: 151 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 152, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 151;
    • i. a heavy chain having the sequence of SEQ ID NO: 153 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 154, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID NO: 153;
    • j. a heavy chain having the sequence of SEQ ID NO: 225 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 226, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID 225; or
    • k. a heavy chain having the sequence of SEQ ID NO: 227 or a variant thereof, and a light chain having the sequence of SEQ ID NO: 228, wherein the variation comprises a knob-and-hole mutation and/or removal of Lys (K) at the C-terminal end in SEQ ID 227.


In preferred embodiments, the ABP comprises two Fc moieties. In some embodiments, the IL-21Rα mutein is linked to the first of the two Fc moieties, and the IL-21 domain is linked to the second of the two Fc moieties. In some embodiments, the IL-21Rα mutein is linked to the C terminus of the first of the two Fc moieties, and the IL-21 domain is linked to the C terminus of the second of the two Fc moieties. Various methods known in the art can be used to link the IL-21Rα mutein to the first of the two Fc moieties, and the IL-21 domain to the second of the two Fc moieties. In some embodiments, the IL-21 domain and the IL-21Rα mutein are respectively linked through a non-cleavable peptide linker or without a peptide linker. In some embodiments, the non-cleavable peptide linker is G4S linker having the sequence of SEQ ID NO: 17. In some embodiments, a non-peptide linker is used. In some embodiments, the non-cleavable peptide linker has a sequence selected from SEQ ID NOs: 212-224.


In some embodiments, the ABP comprises an Fc moiety of a human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc moiety comprises any one sequence selected from SEQ ID NOs: 16, and 185-190. In some embodiments, the Fc moiety comprises an CH3 domain of a human IgG1, IgG2, IgG3 or IgG4.


In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP is a Fv fragment, a Fab fragment, a F(ab′)2 fragment, a Fab′ fragment, a scFv (sFv) fragment, and a scFv-Fc fragment.


In some embodiments, the ABP comprises a knob variant and a hole variant of Fc fragment.


6.5.2. IL-21 Domain


In some embodiments, the IL-21 domain is a human IL-21. In some embodiments, the IL-21 domain is a functional fragment of human IL-21, which can bind to IL-21Rα and activate the target cell. In some embodiments, the IL-21 domain is a functional variant or a homolog of human IL-21, which can bind to IL-21Rα and activate the target cell.


In some embodiments, the IL-21 domain has the sequence of SEQ ID NO: 100 (human IL-21). In some embodiments, the IL-21 domain has a sequence at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 100.


6.5.3. Immunocytokine Structure


In some embodiments, the immunocytokine comprises four polypeptide chains-two identical light chains and two heavy chains, joined to form a heterodimer by knobs-into-holes (KiH) interaction. In some embodiments, one of the two heavy chains (“first chain”) is fused to a capping moiety (e.g., IL-21Rα mutein) and the other one (“second chain”) is fused to IL-21. In some embodiments, IL-21 and the capping moiety are fused to the heavy chains through a peptide linker. In some embodiments, the peptide linker is a non-cleavable and flexible peptide linker.


In some embodiments, the first chain comprising from the N terminus to C terminus:

    • a. a first Fc moiety of the ABP; and
    • b. an IL-21Rα mutein.


In some embodiments, the first chain further comprises a linker between the first Fc moiety of the ABP and the IL-21Rα mutein.


In some embodiments, the first Fc moiety is a human IgG1, IgG2, IgG3 or IgG4 having any one sequence selected from SEQ ID NOs: 16, 185-190. In some embodiments, the first Fc moiety comprises an CH3 domain of a human IgG1, IgG2, IgG3 or IgG4.


In some embodiments, the first chain comprising from the N terminus to C terminus:

    • a. a first heavy chain of the ABP; and
    • b. an IL-21Rα mutein.


In some embodiments, the first chain further comprises a linker between the first heavy chain of the ABP and the IL-21Rα mutein.


In some embodiments, the first heavy chain of the ABP comprises a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227 or a variation thereof. In some embodiments, the variation comprises a knob-and-hole mutation in a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. In some embodiments, the variation comprises removal of Lys (K) at the C-terminal end in a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. In some embodiments, the variation comprises a knob-and-hole mutation and removal of Lys (K) at the C-terminal end in a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 151, 153, 225 and 227. The knob-and-hole mutation can be a mutation for making a knob variant or for making a hole variant for knob-and-hole interaction. In some embodiments, the first heavy chain of the ABP comprises the sequence of SEQ ID NO: 103.


In some embodiments, the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169.


In some embodiments, the first chain comprises a sequence selected from SEQ ID NOs: 104-150 and 192-209.


In some embodiments, the first chain comprises a sequence selected from SEQ ID NOs: 170-184.


In some embodiments, the second chain comprises from the N terminus to C terminus:

    • a. a second Fc moiety of the ABP; and
    • b. an IL-21 domain.


In some embodiments, the second chain further comprises a linker between the second Fc moiety of the ABP and the IL-21 domain.


In some embodiments, the second Fc moiety is a second heavy chain of the ABP.


In some embodiments, the immunocytokine comprises a first heavy chain and a second heavy chain of the ABP. In some embodiments, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation for knob-and-hole interaction. In some embodiments, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation for knob-and-hole interaction.


In some embodiments, the second chain has the sequence of SEQ ID NO: 101.


In some embodiments, the immunocytokine comprises two identical light chains. In some embodiments, the light chain has the sequence of SEQ ID NO: 102. In some embodiments, the light chain is the light chain of any one of the ABP is selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tremelimumab, tiragolumab, relatlimab, or a functional variant thereof.


6.6. Polynucleotide, Vector and Host Cells


One aspect of the present disclosure provides one or more polynucleotides encoding the immunocytokine. In some embodiments, the one or more polynucleotides comprise:

    • a. a first polynucleotide segment encoding a first chain comprising the heavy chain of the ABP and the IL-21Rα mutein;
    • b. a second polynucleotide segment encoding a second chain comprising the heavy chain of the ABP and the IL-21 domain; and
    • c. a third polynucleotide segment encoding the light chain of the ABP.


In some embodiments, the first polynucleotide segment comprises a coding sequence of a first chain comprising the heavy chain of the ABP, a peptide linker and the IL-21Rα mutein. In some embodiments, the first polynucleotide segment comprises a coding sequence of a polypeptide having a sequence selected from SEQ ID NOs: 104-150 and 192-209.


In some embodiments, the second polynucleotide segment comprises a coding sequence of a second chain comprising the heavy chain of the ABP, a peptide linker and the IL-21 domain. In some embodiments, the second polynucleotide segment comprises a coding sequence of a polypeptide having the sequence of SEQ ID NO: 101.


In some embodiments, the third polynucleotide segment comprises a coding sequence of a light chain having the sequence of SEQ ID NO: 102.


In some embodiments, the first polynucleotide segment comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 98% or 99% identity to SEQ ID NO: 210. In some embodiments, the first polynucleotide comprises a sequence of SEQ ID NO: 210 with one or more nucleotide differences corresponding to the one or more amino acid substitutions in IL21RαMutein.


In some embodiments, the first polynucleotide segment comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 98% or 99% identity to SEQ ID NO: 211. In some embodiments, the first polynucleotide comprises a sequence of SEQ ID NO: 211 with one or more nucleotide differences corresponding to the one or more amino acid substitutions in IL21RαMutein.


In some embodiments, the one or more polynucleotides have a sequence which has been codon optimized for expression in a mammalian cell. In some embodiments, the one or more polynucleotides have a sequence which has been codon optimized for expression in a human cell.


In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are in a single polynucleotide molecule. In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are in multiple polynucleotide molecules.


When more than one polynucleotide segments are present in a single polynucleotide molecule, the multiple polynucleotide segments can be separated by internal ribosome entry site (IRES). In some embodiments, the multiple polynucleotide segments are separated by a self-cleavage site.


In some embodiments, the one or more polynucleotides further comprise a regulatory sequence operably linked to the first, second, or third polynucleotide segment. In some embodiments, the one or more polynucleotides comprise more than one regulatory sequences. In some embodiments, the one or more polynucleotides comprise a regulatory sequence for each of the first, second and third polynucleotide segment.


In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are individually present in separate polynucleotide molecules.


In another aspect, the present disclosure provides one or more vectors comprising the one or more polynucleotides. In some embodiments, the first polynucleotide segment, the second polynucleotide segment, and the third polynucleotide segment are individually present in separate vectors. In some embodiments, two or more of the polynucleotide segments are cloned in a single vector.


In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector or a lentiviral vector. In some embodiments, the vector is non-viral. In some embodiments, the vector is a plasmid.


In some embodiments, the one or more polynucleotides or the one or more vectors are present in a host cell. Accordingly, one aspect of the present disclosure provides a host cell comprising the one or more polynucleotides or the one or more vectors. In some embodiments, the host cell expresses the immunocytokine. In some embodiments, the host cell comprises the immunocytokine. In some embodiments, the host cell releases the immunocytokine. In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell.


The host cell can be a eukaryotic cell, for example a fungal cell such as yeast. The host cell can be a mammalian cell (which may be a cell in cell culture, or a cell present in a tissue or organ). In some embodiments, the host cell is a human, mouse, rat, rabbit, bovine or dog (or, for example, any other wild, livestock/domesticated animal) cell. In some embodiments, the host cell is a stable cell line cell, or a primary cell, adherent or suspension cell. As examples, the host cell can be a macrophage, osteosarcoma, or CHO, BHK (baby hamster kidney), Bowes human melanoma cell, 911, AT1080, A549, HEK293, or HeLa cell line cell or a mouse primary cell, but not limited thereto. In some embodiments, the host cell is a bacterial cell, such as E. coli.


The eukaryotic cell can be a plant cell (for example a monocotyledonous or dicotyledenous plant cell; typically an experimental, crop and/or ornamental plant cell, for example Arabidopsis, maize); fish (for example Zebra fish; salmon), bird (for example chicken or other domesticated bird), insect (for example Drosophila; bees), Nematoidia or Protista (for example Plasmodium spp or Acantamoeba spp) cell.


In some embodiments, the host cell is used for production of the immunocytokine. In some embodiments, immunocytokine produced from the host cell is purified for therapeutic use. In some embodiments, the host cell is used as therapeutics.


One aspect of the present disclosure provides a polynucleotide encoding the IL-21Rα mutein. In some embodiments, the polynucleotide encoding IL-21Rα mutein having a sequence selected from SEQ ID NOs: 18-99 and 155-169. In some embodiments, the polynucleotide is a viral or non-viral vector. In some embodiments, the polynucleotide further comprises a regulatory sequence operable linked to the coding sequence of IL-21Rα mutein. In another aspect, the present disclosure provides a host cell comprising the polynucleotide encoding the IL-21Rα mutein.


6.7. Method of Treatment


In another aspect, the present disclosure provides a method of administering the immunocytokine or the host cell expressing immunocytokine described above to a subject. In some embodiments, the subject is a cancer patient.


In some embodiments, the administration is effective in enhancing immune response in the subject. In some embodiments, the administration is effective in treating cancer. In some embodiments, the administration is effective in selectively activating an IL-21Rα on a target cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell.


In some embodiments, the immunocytokine or the host cell is administered in an amount sufficient to enhance immune response in the subject. In some embodiments, the immunocytokine or the host cell is administered in an amount sufficient to treat cancer. In some embodiments, the immunocytokine or the host cell is administered in an amount sufficient to selectively activate an IL-21Rα on a target cell.


In some embodiments, the method comprises administration of the immunocytokine, the host cell or a pharmaceutical composition comprising the immunocytokine or the host cell.


6.8. Pharmaceutical Composition


In one aspect, the present disclosure provides a pharmaceutical composition comprising the immunocytokine or the host cell comprising the immunocytokine provided herein.


In some embodiments, the pharmaceutical composition comprises the immunocytokine and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprising a host cell expressing the immunocytokine and a pharmaceutically acceptable carrier.


In some embodiments, the pharmaceutically acceptable carrier is a sterile aqueous solution or dispersion and sterile powder for preparation of a sterile injectable solution or dispersion. In some embodiments, the composition is formulated for parenteral injection. The composition can be formulated as a solid, a solution, a microemulsion, a liposome, or other ordered structures suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), and suitable mixtures thereof. In some cases, the composition contains an isotonic agent, for example, sugar, polyalcohol, for example, sorbitol or sodium chloride.


In some embodiments, the pharmaceutical composition is provided in a unit dose for use as described above.


7. EXAMPLES

7.1. Generation of IL21Rα Muteins


Nine (9) amino acid residues of IL-21Rα (M70, A71, D72, D73, Y36, E38, L39, I74, and L94) were predicted to form a binding site to IL-21 based on the predicted structure of IL-21 and IL-21Rα. Some amino acid residues (e.g., Q35) of IL-21Rα were additionally predicted to be involved in the binding affinity from the in-silico analysis (Discovery studio). Their roles in binding to IL-21 were further studied by alanine scanning mutagenesis of each of the amino acid residues of IL-21Rα. IL-21RαMuteins were designed by single amino acid substitution to the 20 amino acid residues in the IL-21Rα amino acid sequence as provided in Table 2.









TABLE 2







WT and IL-21RαMuteins








No.
Point mutation











1
WT


2
Y10A


3
Q35K


4
Q35R


5
Q35Y


6
Y36A


7
Y36C


8
Y36E


9
Y36G


10
Y36H


11
Y36I


12
Y36K


13
Y36M


14
Y36N


15
Y36P


16
Y36Q


17
Y36R


18
Y36S


19
Y36T


20
Y36V


21
E38A


22
E38C


23
E38K


24
E38R


25
E38Y


26
L39A


27
L39C


28
L39E


29
L39F


30
L39H


31
L39K


32
L39R


33
L39W


34
L39Y


35
F67A


36
H68A


37
M70C


38
M70D


39
M70F


40
M70G


41
M70H


42
M70K


43
M70L


44
M70N


45
M70Q


46
M70R


47
M70S


48
M70T


49
M70V


50
M70W


51
M70Y


52
A71E


53
A71F


54
A71I


55
A71L


56
A71Q


57
A71R


58
A71W


59
A71Y


60
D72A


61
D72C


62
D72E


63
D72G


64
D72H


65
D72K


66
D72Q


67
D72R


68
D72W


69
D72Y


70
D73A


71
I74A


72
I74K


73
I74R


74
I74W


75
L94A


76
L94F


77
L94K


78
L94Q


79
L94R


80
L94Y


81
P126A


82
Y129A


83
M130A


84
K134A


85
S189A


86
S190A


87
Y191A









The IL21R muteins were generated by introducing one or more point mutations to a plasmid encoding wild type IL-21Rα. Human IgG1Fc (Pro100-Lys330) and IL21R α (Cys20-Glu232) wild type or muteins were conjugated by (G4S)3 linker. Azurocidin signal peptide was added at the N-terminal for secretion of the expressed protein. After verification of the constructs by sequencing, a large-scale plasmid preparation was performed to obtain enough DNA for transfection.


7.2. SPR Full Kinetics Assay of IL-21Rα Muteins against IL-21


Bivalent Fc-fusion proteins (IgG1) were generated with each of the muteins [IL21RαMutein-Fc] and their binding affinity to IL-21 was measured by SPR (Biacore 8K) (Table 3 and FIGS. 2A-2V). IL21Rα Muteins and IL-21's affinity was tested by CM5 sensor chip. 400 mM EDC and 100 mM NHS (Cytiva) were injected to CMS sensor chip for 420 s with a flow rate of 10 μL/min as activator prior to injecting 1.55 ug/mL of hIL-21 in 10 mM NaAc (pH 5.0) to the channel for 240 s at a flow rate of 10 μL/min. The chip was deactivated by 1M ethanolamine-HCl (Cytiva) at flow rate of 10 μL/min for 420 s.


Multiple cycle kinetics were used to perform the assay. hIL-21R (WT or Muteins) at 7 different concentrations and a running buffer were injected orderly to Fc1-Fc2 at a flow rate of 80 μL/min for an association phase of 120 s, followed by 1000 s dissociation. 10 mM glycine pH1.5 was injected as a regeneration buffer following every dissociation phase.


The sensorgrams from the reference channel Fc1 and the buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1:1 binding model or heterogeneous ligand. Molecular weight of 15 kDa were used to calculate the molar concentration of IL-21.


The data from the SPR full kinetics assay of IL-21Rα muteins against IL21 are provided in FIGS. 2A-2V. The binding affinities of muteins were measured using Biacore 8K and provided in Table 3.









TABLE 3







Binding affinity of Fc-IL21Rα (WT


and mutein) against IL21 (1:1 binding model)









No.
Point Mutation
KD (M)












1
WT
1.67E−10


2
Y10A
5.23E−10


3
Q35K
3.43E−10


4
Q35R
4.91E−10


5
Q35Y
3.66E−10


6
Y36A
2.13E−09


7
Y36C
1.10E−09


8
Y36E
6.88E−10


9
Y36G
2.76E−09


10
Y36H
6.11E−10


11
Y36I
2.55E−09


12
Y36K
2.97E−09


13
Y36M
7.26E−10


14
Y36N
8.94E−10


15
Y36P
2.46E−07


16
Y36Q
1.04E−09


17
Y36R
6.67E−09


18
Y36S
2.58E−09


19
Y36T
3.98E−09


20
Y36V
3.33E−09


21
E38A
2.85E−08


22
E38C
2.40E−08


23
E38K
>1.00E−06*


24
E38R
>1.00E−06 


25
E38Y
1.77E−07


26
L39A
2.36E−08


27
L39C
1.50E−07


28
L39E
1.02E−07


29
L39F
2.65E−10


30
L39H
1.98E−09


31
L39K
1.41E−08


32
L39R
9.70E−08


33
L39W
2.33E−09


34
L39Y
8.16E−10


35
F67A
4.37E−10


36
H68A
1.68E−10


37
M70C
>1.00E−06 


38
M70D
>1.00E−06 


39
M70F
1.18E−09


40
M70G
>1.00E−06 


41
M70H
6.94E−07


42
M70K
>1.00E−06 


43
M70L
6.42E−10


44
M70N
1.16E−07


45
M70Q
6.85E−07


46
M70R
>1.00E−06 


47
M70S
8.54E−08


48
M70T
5.12E−09


49
M70V
9.74E−10


50
M70W
7.06E−07


51
M70Y
7.92E−08


52
A71E
2.92E−09


53
A71F
1.01E−09


54
A71I
1.97E−09


55
A71L
1.26E−09


56
A71Q
5.01E−09


57
A71R
4.09E−07


58
A71W
1.85E−08


59
A71Y
1.03E−08


60
D72A
>1.00E−06 


61
D72C
>1.00E−06 


62
D72E
1.14E−06


63
D72G
>1.00E−06 


64
D72H
>1.00E−06 


65
D72K
>1.00E−06 


66
D72Q
>1.00E−06 


67
D72R
>1.00E−06 


68
D72W
>1.00E−06 


69
D72Y
>1.00E−06 


70
D73A
>1.00E−06 


71
I74A
7.66E−10


72
I74K
5.18E−10


73
I74R
1.30E−09


74
I74W
1.15E−09


75
L94A
1.44E−09


76
L94F
1.49E−09


77
L94K
2.79E−07


78
L94Q
1.19E−09


79
L94R
3.97E−08


80
L94Y
1.13E−09


81
P126A
2.77E−10


82
Y129A
5.15E−10


83
M130A
2.54E−10


84
K134A
6.18E−10


85
S189A
2.90E−10


86
S190A
2.45E−10


87
Y191A
6.67E−10





*1.00E−06 is the minimum detection limit.






After the measurement of binding affinities of the muteins [IL21Rα(mut)-Fc] to IL-21, 58 muteins were classified based on the degree of reduction in their binding affinity to IL-21, e.g., 10, 100, and 1000-fold reduction compared to wild-type IL-21Rα. Finally, 66 IgG-fusion proteins were generated in which an IL-21 and one of the muteins of IL21-Rα are fused to one of two heavy chains of IgG, respectively.


7.3. Generation of Immunocytokines (αPD-1IL21RαMutein/IL21)


The immunocytokine described herein, αPD-1IL21RαMutein/IL21, can exhibit an anti-cancer immune response by working as an ICB and inducing signal transduction mediated by the complex of IL21 receptor (IL21Rα/common gamma chain) expressed on the surface of target cells. αPD-1IL21RαMutein/IL21 is designed to primarily activate target immune cells only when it binds to PD-1. It leads competition between IL21RαMutein and endogenous IL21Rα (e.g., IL21RαWT) of target cells by the proximity, inducing stripping of IL21RαMutein from the moiety of IL-21, causing it to bind to endogenous IL21Rα.


Previously, a fusion protein comprising an attenuated IL-21 fused to the c-terminal ends of the anti-PD-1 antibody was developed for treatment of cancer by activation of immune cells. In the fusion protein, an attenuated IL-21 was used to reduce off-target effects and the anti-PD-1 antibody was used to improve bioavailability at the target. The attenuated IL-21 includes two point mutations in the amino acid sequence of IL-21, making its max potency reduced to 70-80% compared to wild-type IL-21 (See Shanling Shen et al. Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity. Front Immunol. 2020 May 8; 11:832).


Unlike the fusion protein comprising an attenuated IL-21, αPD-1IL21RαMutein/IL21 includes an unmodified IL-21, thus they can have effects on target cells similar to wild-type IL-21. 66 immunocytokines, each containing a different mutein of IL21Rα, were generated. The 66 immunocytokines (Table 4) include one or two amino acid substitutions. More specifically, the immunocytokines (R-kine-1 to 66) includes (i) a first chain comprising a heavy chain, G4S linker and IL-21RαMutein; (ii) a second chain comprising a heavy chain, G4S linker and a human IL-21; and (iii) two light chains, as specified in Table 4.













TABLE 4







Sequence of first
Sequence of second




IL-21Rα
chain (Heavy
chain (Heavy



Mutation
Chain-G4S Linker-
Chain-G4S Linker-
Sequence of two


No.
Site
IL-21RαMutein)
human IL-21)
light chains







R-kine-1
WT
SEQ ID NO: 191
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-2
Y36C
SEQ ID NO: 104
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-3
Y36E
SEQ ID NO: 105
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-4
Y36G
SEQ ID NO: 106
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-5
Y36H
SEQ ID NO: 107
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-6
Y36I
SEQ ID NO: 108
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-7
Y36K
SEQ ID NO: 109
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-8
Y36M
SEQ ID NO: 110
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-9
Y36N
SEQ ID NO: 111
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-10
Y36P
SEQ ID NO: 112
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-11
Y36Q
SEQ ID NO: 113
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-12
Y36R
SEQ ID NO: 114
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-13
Y36S
SEQ ID NO: 115
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-14
Y36T
SEQ ID NO: 116
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-15
Y36V
SEQ ID NO: 117
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-16
E38C
SEQ ID NO: 118
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-17
E38R
SEQ ID NO: 200
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-18
E38Y
SEQ ID NO: 119
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-19
L39C
SEQ ID NO: 120
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-20
L39E
SEQ ID NO: 121
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-21
L39H
SEQ ID NO: 122
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-22
L39K
SEQ ID NO: 123
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-23
L39R
SEQ ID NO: 124
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-24
L39W
SEQ ID NO: 125
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-25
L39Y
SEQ ID NO: 126
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-26
M70F
SEQ ID NO: 127
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-27
M70H
SEQ ID NO: 128
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-28
M70N
SEQ ID NO: 129
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-29
M70Q
SEQ ID NO: 130
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-30
M70S
SEQ ID NO: 131
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-31
M70T
SEQ ID NO: 132
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-32
M70V
SEQ ID NO: 133
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-33
M70W
SEQ ID NO: 134
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-34
M70Y
SEQ ID NO: 135
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-35
A71E
SEQ ID NO: 136
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-36
A71F
SEQ ID NO: 137
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-37
A71I
SEQ ID NO: 138
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-38
A71L
SEQ ID NO: 139
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-39
A71Q
SEQ ID NO: 140
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-40
A71R
SEQ ID NO: 141
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-41
A71W
SEQ ID NO: 142
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-42
A71Y
SEQ ID NO: 143
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-43
I74R
SEQ ID NO: 144
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-44
I74W
SEQ ID NO: 145
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-45
L94F
SEQ ID NO: 146
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-46
L94K
SEQ ID NO: 147
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-47
L94Q
SEQ ID NO: 148
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-48
L94R
SEQ ID NO: 149
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-49
L94Y
SEQ ID NO: 150
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-50
M70C
SEQ ID NO: 201
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-51
M70D
SEQ ID NO: 202
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-52
M70G
SEQ ID NO: 203
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-53
M70R
SEQ ID NO: 204
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-54
D72A
SEQ ID NO: 205
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-55
D72E
SEQ ID NO: 206
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-56
D72Q
SEQ ID NO: 207
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-57
D72R
SEQ ID NO: 208
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-58
D73A
SEQ ID NO: 209
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-59
Y36A + D72E
SEQ ID NO: 192
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-60
Y36A + L94R
SEQ ID NO: 193
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-61
E38A + D72E
SEQ ID NO: 194
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-62
E38A + L94K
SEQ ID NO: 195
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-63
E38A + L94R
SEQ ID NO: 196
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-64
E38R + D72R
SEQ ID NO: 197
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-65
D72E + L94K
SEQ ID NO: 198
SEQ ID NO: 101
SEQ ID NO: 102


R-kine-66
D72E + L94R
SEQ ID NO: 199
SEQ ID NO: 101
SEQ ID NO: 102









For production of the immunocytokines, 6.0×106/mL of ExpiCHO cells (ThermoFisher) with higher than 95% viability were prepared in 100 mL of cell culture media. 100 μg of the plasmid DNA encoding the immunocytokine was mixed with the ExpiFectamine™ CHO transfection reagent (ThermoFisher) and the mixture was added to the cell culture media. The cell culture was incubated in a platform shaker with the rotation rate at 150 rpm. The temperature was maintained at 37° C. while CO2 level at 8%.


After ten days of incubation, the cells were pelleted by centrifuging at 4000 rpm, 25° C. for 10 minutes. Supernatant was collected for purification and gel electrophoresis. The supernatant was loaded on SDS-PAGE gel, following the instruction for NuPAGE™ 4-12% Bis-Tris Protein Gels (ThermoFisher). PageRuler™ Unstained Protein Ladder (ThermoFisher) was used alongside with the protein samples to determine the molecular weight of the protein. Fusion proteins were then purified by Protein A column (Cytiva) followed by SEC column (Cytiva).


7.4. Homogeneous Time-Resolved Fluorescence (HTRF) Phospho-STAT3 Assay of Immunocytokines (αPD-1IL21RαMutein/IL21)


The 66 immunocytokines were evaluated by measuring phosphorylation of STAT3 in HTRF-based high-throughput assay. Human cutaneous T lymphocyte cell lines (H9 (Cobioer), derivative of Hut78 cells) and H9 cells that stably expressing a programmed cell death protein 1 (PD-1(+) H9) were used in the pSTAT3 assay. Cells were grown in IMDM medium (Gibco) containing 20% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Sigma Aldrich) for H9. 3 μg/mL puromycin (Invivogen) was additionally added for PD-1 positive H9 cells. Subculture of cells was conducted every 48 hours to avoid high density which could arrest the cell cycle.


Measuring pSTAT3 production was conducted to investigate the activation of cells by IL-21 binding with IL-21 receptors and common gamma chains. The high production of pSTAT3 was considered as a marker of strong reaction of treated materials. To conduct experiments, pSTAT3 ELISA kit (Perkin Elmer, MA) and Flex Station 3 (Molecular Devices, CA) were used following the manufacturer's user guide.


The detailed description is as follows. PD-1(−) H9 or PD-1(+) H9 cells were incubated with serum free media on overnight. After incubation, spin-down cells (with 125 g) were harvested with HBSS (Gibco) solution and seeded on white 96 well low volume plate (Cisbio) by 2.5×104 cells/well/8 μL. Compounds for evaluation were prepared with 3× concentration of final concentration and treated to cells for 30 minutes at 37° C. The lysis buffer was added to the wells for 30 minutes and then reagents for HTRF reaction were treated following the manufacturer's protocol. After 24 hours, the HTRF reaction was measured by Flex Station 3 equipment.


The non-linear analysis (4 parameters logistic regression) was conducted to calculate experiment parameters including EC50, Maximal response, and Hillslope. The Black and Leff operational model was adopted to estimate the compound's intrinsic efficacy.



FIGS. 4, 5, and 6A-6E show that the moiety of an anti-PD-1 antibody of αPD-1IL21RαMutein/IL21 contributed to differences in the pharmacodynamics of αPD-1IL21RαMutein/IL21 on PD-1(+) or PD-1(−) H9 cells.


To be specific, the value of EC50 of phosphorylation of STAT3 observed in αPD-1IL21RαMutein/IL21 treated PD-1(−) cells were higher than PD-1(+) cells, and the max efficacy was similar in both cell lines (Table 5).









TABLE 5







Summary of EC50










PD-1(−) H9 cells
PD-1(+) H9 cells











Mutation
EC50 (M)
sem
EC50 (M)
sem





Wildtype
2.58E−07
4.53E−08
1.77E−07
3.78E−08


D72E
1.57E−07
2.03E−08
3.29E−09
5.80E−10


A71R
1.29E−07
2.57E−08
4.87E−09
6.83E−10


Y36G
1.44E−07
3.80E−08
1.28E−08
3.68E−09


M70Q
1.75E−07
2.55E−08
6.14E−09
6.72E−10


M70H
1.98E−07
3.57E−08
6.93E−09
8.64E−10


M70W
1.90E−07
2.99E−08
6.66E−09
9.50E−10


L94K
2.53E−07
5.90E−08
1.03E−08
1.88E−09


E38R
2.13E−07
4.37E−08
1.30E−09
1.49E−10


M70R
1.63E−08
3.00E−09
3.21E−10
4.36E−11


M70D
1.51E−07
3.17E−08
1.06E−09
1.08E−10


M70C
2.96E−07
6.20E−08
1.15E−07
2.63E−08


M70G
4.19E−07
8.23E−08
2.91E−09
4.02E−10


D72R
1.20E−08
2.60E−09
5.04E−10
6.72E−11


D72Q
1.65E−07
3.35E−08
9.50E−10
1.17E−10


D72A
1.39E−07
1.59E−08
1.22E−09
1.71E−10


D73A
6.45E−08
9.86E−09
6.13E−10
8.81E−11


D72E + E38A
2.69E−08
6.03E−09
4.38E−10
7.87E−11









From the results of HTRF-based high-throughput screening, six variants of αPD-1IL21RαMutein/IL21, each containing a different mutein selected from E38R, M70D, M70H, M70Q, D72A, and L94K, were selected for further study (FIG. 3). Among these six variants, M70Q and M70H muteins showed efficacy comparable to wild type IL-21, distinguishing from a fusion protein containing an attenuated IL-21 mentioned above. The attenuated IL-21 showed less than 80% efficacy compared to wild type IL-21 (FIGS. 4 and 5).


These results demonstrate that IL-21Rα muteins of αPD-1IL21RαMutein/IL21 act as a capping molecule inhibiting IL-21 from binding to non-target cells, which is the reason for the low signal intensity in PD-1(−) cells. This shows that αPD-1IL21RαMutein/IL21 is an immunocytokine having high tissue specificity.


Besides, increasing specificity while maintaining efficacy of the drug substance by introducing a proper modification to a capping moiety to adjust specificity to its receptor is a unique advantage of our invention distinguishing from other drugs. αPD-1IL21RαMutein/IL21 shows characteristics of both full agonist and competitive antagonist.


7.5. SPR Full Kinetics Assay of Immunocytokines (αPD-1IL21Rα Mutein/IL21) against PD-1


Interaction between immunocytokine and human PD-1 (hPD-1) was determined by Surface Plasmon Resonance (SPR, Biacore 8K) analysis. Immunocytokines and hPD-1's affinity was tested by CMS sensor chip. 400 mM EDC and 100 mM NHS (Cytiva) were injected to CM5 sensor chip for 420 s with a flow rate of 10 μL/min as activator prior to injecting 25 μg/mL of anti-human Fc IgG in 10 mM NaAc (pH 4.5) to the channel 1-8 for 420 s at a flow rate of 10 μL/min. The chip was deactivated by 1M ethanolamine-HCl (Cytiva) at flow rate of 10 μL/min for 420 s.


Immunocytokines diluted in running buffer (1× HBS-EP+) were captured on to Fc2 via anti-human Fc IgG at flow rate of 10 μL/min for 40 s. Multiple cycle kinetics was used to perform the assay. The analyte hPD-1 at 7 different concentrations (0, 2.5, 5, 10, 20, 40, and 80 nM) and running buffer were injected orderly to Fc1-Fc2 at a flow rate of 30 μL/min for an association phase of 180 s, followed by 900 s dissociation. 10 mM glycine pH 1.5 was injected as regeneration buffer following every dissociation phase.


The sensorgrams from the reference channel Fc1 and the buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1:1 binding model. Molecular weight of 17 kDa were used to calculate the molar concentration of hPD-1.


The SPR analysis demonstrated that the fusion of IL21RαWT or IL21RαMutein and IL-21 to the anti-PD-1 antibody did not affect the affinity of the anti-PD-1 antibody to PD-1 (FIG. 7A-7Q, Table 6).











TABLE 6





No.
IL-21R Mutation Site
KD (M)

















1
WT
8.41E−09


2
Y36C
8.36E−09


3
Y36E
8.32E−09


4
Y36G
8.51E−09


5
Y36H
9.02E−09


6
Y36I
8.74E−09


7
Y36K
8.23E−09


8
Y36M
8.32E−09


9
Y36N
8.20E−09


10
Y36P
8.44E−09


11
Y36Q
8.49E−09


12
Y36R
8.45E−09


13
Y36S
8.39E−09


14
Y36T
8.52E−09


15
Y36V
8.63E−09


16
E38C
9.06E−09


17
E38R
6.61E−09


18
E38Y
8.73E−09


19
L39C
8.61E−09


20
L39E
8.45E−09


21
L39H
8.24E−09


22
L39K
8.43E−09


23
L39R
8.62E−09


24
L39W
8.78E−09


25
L39Y
9.74E−09


26
M70F
9.01E−09


27
M70H
8.85E−09


28
M70N
8.78E−09


29
M70Q
8.85E−09


30
M70S
8.84E−09


31
M70T
8.49E−09


32
M70V
8.53E−09


33
M70W
8.86E−09


34
M70Y
8.81E−09


35
A71E
8.14E−09


36
A71F
8.70E−09


37
A71I
8.59E−09


38
A71L
8.87E−09


39
A71Q
8.51E−09


40
A71R
8.94E−09


41
A71W
9.12E−09


42
A71Y
8.94E−09


43
I74R
8.90E−09


44
I74W
8.94E−09


45
L94F
8.39E−09


46
L94K
8.90E−09


47
L94Q
8.90E−09


48
L94R
7.82E−09


49
L94Y
9.19E−09


50
M70C
7.91E−09


51
M70D
7.25E−09


52
M70G
8.42E−09


53
M70R
6.83E−09


54
D72A
6.88E−09


55
D72E
6.53E−09


56
D72Q
8.64E−09


57
D72R
8.03E−09


58
D73A
7.47E−09


59
Y36A + L94R
7.26E−09


60
E38A + L94K
7.70E−09


61
E38A + L94R
8.16E−09


62
D72E + Y36A
7.39E−09


63
D72E + E38A
7.19E−09


64
D72E + L94K
7.58E−09


65
D72E + L94R
8.01E−09


66
D72R + E38R
7.02E−09









7.6. Binding Affinity of Immunocytokines (αPD-1IL21RαMutein/IL21) to FcRn


The binding affinity of antibody-based protein drugs to FcRn is known to be highly associated with its half-life in vivo. The binding affinity of immunocytokines (αPD-1IL21RαMutein/IL21) to FcRn was measured using Bio-Layer Interferometry (BLI) system. As a control, the binding affinity of anti-PD-1 antibody which is not conjugated to IL-21 or IL-21 RαMutein was also measured.


For the assay, FAB2G biosensor (Sartorius) was hydrated with a running buffer for 10 minutes in the 96 well plate (Corning). The ligands (anti-PD-1 antibody or Immunocytokine) were diluted with the running buffer to make a final concentration of 0.5 μg/ml for anti-PD-1 antibody and 2 μg/ml for immunocytokine. FAB2G biosensor was loaded with either anti-PD-1 antibody or Immunocytokine at 1.5nm level. After loading either anti-PD-1 antibody or Immunocytokine, the baseline was set by incubating the loaded sensor tip in the running buffer for 300 sec. Ligand loaded sensor tips were incubated in wells containing a 2-fold serial dilution of soluble, FcRn/B2M complex receptors. Association and dissociation were measured for 60 seconds or until a steady state was reached. The measurement data are provided in FIG. 8A.


The binding affinities of the anti-PD-1 antibody or Immunocytokine to FcRn were measured using Octet RED96e (ForteBio) instruments. Optimized Octet sample buffer (100 mM Sodium Phosphate, 300 mM NaCl, 0.05% Tween20) was used for sample dilution and all binding baseline, association, and dissociation steps at either pH of 6.0 or pH of 7.4. A buffer only blank curve was subtracted to correct any drift. The data were fit to a 1:1 binding model using ForteBio data analysis software 11.1 to determine the Kon, Koff, and KD, which are provided in FIG. 8B.


The data show that the binding affinity of the immunocytokine to FcRn is not significantly different from the binding affinity of the anti-PD-1 antibody. This result suggests that the pharmacokinetic profile of the instant immunocytokine will benefit from FcRn binding ability, thus having a half-life sufficient to provide therapeutic effects.


7.7. In Vitro Tumor Killing Assay


To confirm the anti-tumor effects of the present immunocytokine (αPD-1IL21RαMutein/IL21), an increase in IFNγ expression level and a change in cytotoxicity of the CD8+ T cells that are treated with the present immunocytokine were tested. When the CD8+ T cells are co-cultured with autologous monocyte-derived DCs (moDCs) presenting specific antigens on their surfaces through MHC-peptide complexes, the tumor antigen educated CD8+ T cells (e.g., CTLs) can recognize and attack tumor cells expressing those antigens. The efficacy of the immunocytokines was confirmed by measuring fluorescent materials leaked from the tumor cells due to the death of tumor cells.


Specifically, human PBMCs were purchased from StemExpress (USA). Monocytes were isolated using Pan Monocyte Isolation Kit (Miltenyi Biotec) and were cultured for 7 days with 35 ng/mL recombinant human IL-4 (R&D Systems) and 50 ng/mL GM-CSF (R&D Systems) in RPMI1640 medium(Gibco) to differentiate the monocyte to dendritic cells (DCs). The premature monocyte-derived DCs were further matured for 3 days using 10 ng/mL recombinant human IL-6 (R&D Systems), 15 ng/mL IL-1β (R&D Systems), 40 ng/mL TGFα (R&D Systems), and 1 μg/mL PGE2 (PeproTech). During maturation, antigen peptides were loaded on the monocyte-derived DCs (moDCs). Autologous donor's CD8+ T cells were isolated using CD8+ T Cell Isolation Kit (Miltenyi Biotec) and were co-cultured with the matured moDCs for 10 days at a 10:1 cell number ratio. Culture medium supplemented with recombinant human IL-15 (R&D Systems) and recombinant human IL-7 (R&D Systems) were added every 2 or 3 days to sustain CTLs.


CTLs were then expanded using an anti-CD3ε antibody (R&D Systems), anti-CD28 antibody (R&D Systems), and recombinant human IL-2 (R&D Systems) for 5 days. During the expansion of CTLs (effector cell), the present immunocytokines (αPD-1IL21RαMutein/IL21 or αPD-1IL21RαWT/IL21) or controls (e.g., anti-PD-1 antibody) were treated at 500nM concentration.


7.7.1. Release of IFN-γ


IFNγ levels in the culture supernatants were measured by ELISA using Human IFN-gamma DuoSet ELISA kit (R&D Systems). The results are provided in FIG. 10, confirming increased IFNγ release from CTL in response to immunocytokines (four variants of αPD-1IL21RαMutein/IL21, each containing a different mutein selected from M70D, M70Q, L94K, and E39R).


7.7.2. Cytotoxicity


To confirm tumor killing efficacy, Calcein AM(Invitrogen)-stained target cells (MeWo cell line or CMV pp65 gene transduced A375 cell line (A375_CMV)) were plated the day before co-culture with the expanded CTLs (effector cells). The effector cells were collected and loaded to the medium with target cells and cultured for 36 hours. The release of Calcein AM from the dead tumor cells were measured by detecting fluorescent signals at Ex 485 nm and Em 530 nm using FlexStation3 equipment.



FIGS. 11A and 11B provide data from MeWo cell line and A375_CMV cell line, respectively. The data show that CTLs treated with αPD-1IL21RαMutein/IL21 showed better tumor-killing activity than the controls. This can be due to enhancement of effector function of CTLs by αPD-1IL21RαMutein/IL21. These suggest that immunocytokines provided here, αPD-1IL21RαMutein/IL21, can enhance anti-tumor activity when applied to cancer patients.


7.8. Immunocytokines against CTLA-4, TIGIT, LAG-3 (αCTLA-4L21RαMutein/IL21; αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21)


Two immunocytokines against each of three different targets, CTLA-4, TIGIT, and LAG-3 (αCTLA-4L21RαMutein/IL21; αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21) were generated by methods described above related to αPD-1L21RαMutein/IL21. The immunocytokines includes (i) a first chain comprising a heavy chain, G4S linker and IL-21RαMutein; (ii) a second chain comprising a heavy chain, G4S linker and a human IL-21; and (iii) two light chains, as specified in Table 7. The immunocytokines were successfully generated from the CHO cell lines, and the HTRF assay confirmed their functional activity of phosphorylation of STAT3 as described in 5.9.














TABLE 7








Sequence of first
Sequence of second




IL-21Rα
chain (Heavy
chain (Heavy



Mutation
Chain-G4S Linker-
Chain-G4S Linker-
Sequence of two



Site
IL-21RαMutein)
human IL-21)
light chains




















Ipilimumab
WT
SEQ ID NO: 230
SEQ ID NO: 229
SEQ ID NO: 152



M70D
SEQ ID NO: 231
SEQ ID NO: 229
SEQ ID NO: 152



D72A
SEQ ID NO: 232
SEQ ID NO: 229
SEQ ID NO: 152


Tiragolumab
WT
SEQ ID NO: 234
SEQ ID NO: 233
SEQ ID NO: 226



M70D
SEQ ID NO: 235
SEQ ID NO: 233
SEQ ID NO: 226



D72A
SEQ ID NO: 236
SEQ ID NO: 233
SEQ ID NO: 226


Relatlimab
WT
SEQ ID NO: 238
SEQ ID NO: 237
SEQ ID NO: 228



M70D
SEQ ID NO: 239
SEQ ID NO: 237
SEQ ID NO: 228



D72A
SEQ ID NO: 240
SEQ ID NO: 237
SEQ ID NO: 228









7.9. Homogeneous Time-Resolved Fluorescence (HTRF) Phosphor-STAT3 Assay of Immunocytokines (αCTLA-4IL21RαMutein/IL21; αTIGITIL21RαMutein/IL21; and αLAG-3IL21RαMutein/IL21)


The immunocytokines against CTLA-4, TIGIT or LAG-3 were evaluated by measuring phosphorylation of STAT3 in HTRF-based high-throughput assay. Human cutaneous T lymphocyte cell lines (H9 (Cobioer), derivative of Hut78 cells) were grown in IMDM medium (Gibco) containing 20% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Sigma Aldrich) for H9. 3 μg/mL puromycin (Invivogen) was additionally added to the H9 cells. Subculture of cells was conducted every 48 hours to avoid high density which could arrest the cell cycle.


H9 cells were incubated with serum free media on overnight. After incubation, spin-down cells (with 125 g) were harvested with HBSS (Gibco) solution and seeded on white 96 well low volume plate (Cisbio) by 2.5×104 cells/well/8 μL. Compounds for evaluation were prepared at 3× of the final concentration and applied to cells for 30 minutes at 37° C. The lysis buffer was added to the wells for 30 minutes and then reagents for HTRF reaction were treated following the manufacturer's protocol. After 24 hours, the HTRF reaction was measured by Flex Station 3 equipment.



FIG. 12 and Table 8 provide data demonstrating that rhIL21, ABP-IL21RαWT/IL21, and ABP-IL21RαMutein/IL21 activated HTRF reaction. Among them, ABP-IL21RαWT/IL21 and ABP-IL21RαMutein/IL21 had significant lower activity than rhIL21, because of the masking effects of IL21RαWT or IL21RαMutein against IL21. As expected given that IL21RαWT has a higher affinity to IL21 compared to IL21RαMutein, the masking effects of IL21RαWT were greater than IL21RαMutein.









TABLE 8





Summary of EC50























αCTLA-4
αCTLA-4
αCTLA-4




anti-CTLA-
IL21Rα
IL21RαMutein
IL21RαMutein



rhIL21
4 antibody
WT/IL21
(M70D)/IL21
(D72A)/IL21





EC50(M)
7.47E−10
N/A
6.524E−07
3.399E−07
5.143E−07


EC50 ratio
1.0
N/A
873.4
455.0
688.5







anti-
αTIGIT
αTIGIT
αTIGIT




TIGIT
IL21Rα
IL21RαMutein
IL21RαMutein




antibody
WT/IL21
(M70D)/IL21
(D72A)/IL21





EC50(M)

N/A
6.096E−07
2.017E−07
1.67E−07


EC50 ratio

N/A
816.1
270.0
223.6








αLAG-3
αLAG-3
αLAG-3




anti-LAG-
IL21Rα
IL21RαMutein
IL21RαMutein




3 antibody
WT/IL21
(M70D)/IL21
(D72A)/IL21





EC50(M)

N/A
2.73E−06
1.42E−07
4.538E−07


EC50 ratio

N/A
3654.6 
190.1
607.5









7.10. SPR Full Kinetics Assay of Immunocytokines (αCTLA-4IL21RαMutein/IL21; αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21)


Binding between the immunocytokines and their respective human target proteins (hCTLA-4, hTIGIT, or hLAG-3) was tested by Surface Plasmon Resonance (SPR) analysis. Affinities of the immunocytokines to their human ligands were tested by CM5 sensor chip. 400 mM EDC and 100 mM NHS (Cytiva) were injected to CM5 sensor chip for 420 s with a flow rate of 10 μL/min as activator prior to injecting 25 μg/mL of anti-human Fc IgG in 10 mM NaAc (pH 4.5) to the channel 1-8 for 420 s at a flow rate of 10 μL/min. The chip was deactivated by 1M ethanolamine-HCl (Cytiva) at flow rate of 10 μL/min for 420 s.


Immunocytokines diluted in running buffer (1× HBS-EP+) were captured on to Fc2 via anti-human Fc IgG at flow rate of 10 μL/min for 40 s. Multiple cycle kinetics was used to perform the assay. 6 concentrations (1.56, 3.13, 6.25, 12.5, 25, and 50 nM) of analyte hCTLA-4 (Acro Biosystems) or 6 concentrations (0.78, 1.56, 3.13, 6.25, 12.5, and 25 nM) of analyte hTIGIT (R&D systems) or 6 concentrations (0.31, 0.63, 1.25, 2.5, 5, and 10nM) of analyte hLAG-3 (Acro Biosystems) and running buffer were injected orderly to Fc1-Fc2 at a flow rate of 30 μL/min for an association phase of 180 s, followed by 900 s dissociation. 10 mM glycine pH 1.5 was injected as a regeneration buffer following every dissociation phase.


The sensorgrams for reference channel Fc1 and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1:1 binding model or heterogeneous ligand model.


The SPR analysis demonstrated that the fusion of IL21RαWT or IL21RαMutein and IL-21 to the anti-CTLA-4, anti-TIGIT or anti-LAG-3 antibody did not affect the affinity of the anti-CTLA-4, anti-TIGIT or anti-LAG-3 antibody to its respective target (Table 9; FIGS. 9A, 9B and 9C).









TABLE 9







Affinity of immunocytokines (αCTLA-4IL21RαMutein/IL21;


αTIGITIL21RαMutein/IL21; or αLAG-3IL21RαMutein/IL21)


against targets (CTLA-4; TIGIT; or LAG-3)













Control


IL-21R



No.
Antibody
KD (M)
No.
Mutation Site
KD (M)













1
Ipilimumab
2.09E−08
Against





hCTLA-4












2
Tiragolumab
5.95E−11
1
WT
1.80E−08


3
Relatlimab
2.39E−10
2
M70D
1.81E−08










N/A
3
D72A
1.71E−08









Against



hTIGIT











4
WT
5.17E−11



5
M70D
5.37E−11



6
D72A
5.60E−11









Against



hLAG-3











7
WT
2.64E−10



8
M70D
2.49E−10



9
D72A
2.55E−10










8. EQUIVALENTS AND INCORPORATION BY REFERENCE

While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.


All references, issued patents and patent applications cited within the body of the instant specification, are hereby incorporated by reference in their entirety, for all purposes.


9. SEQUENCE LISTING












Summary of Sequence Listing








SEQ



ID NO
Sequences





 1
Nivolumab heavy chain


 2
Nivolumab light chain


 3
pembrolizumab heavy chain


 4
pembrolizumab light chain


 5
cemiplimab heavy chain


 6
cemiplimab light chain


 7
atezolizumab heavy chain


 8
atezolizumab light chain


 9
dostarlimab heavy chain


 10
dostarlimab light chain


 11
durvalumab heavy chain


 12
durvalumab light chain


 13
avelumab heavy chain


 14
avelumab light chain


 15
Wild type IL-21Rα (ectodomain; extracellular domain)


 16
Human IgG1 Fc (100 Pro-330 Lys)


 17
G4S linker


18-99
IL-21RαMutein


100
Human IL-21


101
Second Chain (Heavy chain of anti-PD-1 antibody + linker +



human IL-21) with knob mutation


102
Anti-PD-1 antibody, Light chain


103
Anti-PD-1 antibody, Heavy chain with hole mutation


104-150
αPD-1 + linker + IL21RαMutein


151
(Ipilimumab heavy chain)


152
(Ipilimumab light chain)


153
(tremelimumab heavy chain)


154
(tremelimumab light chain)


155-169
IL21RαMutein


170-184
Fc-Linker-IL21RαMutein


185
IgG1 Fc moiety (WT)


186
IgG2 Fc moiety (WT)


187
IgG3 Fc moiety (WT)


188
IgG4 Fc moiety (WT)


189
IGHG1 (Immunoglobulin heavy constant gamma 1) with



‘LALA’(L234A/L235A) mutation


190
IGHG4 (Immunoglobulin heavy constant gamma 4) with



‘SPLE’(S228P/L235E) mutation


191
αPD-1-linker- IL21RαWT


192-209
αPD-1 + linker + IL21RαMutein


210
Polynucleotide encoding IgG1-1IL21Rα (wild type)


211
Polynucleotide encoding αPD-1IL21Rα (wild type)


212-217
Flexible linkers


218-224
Rigid linkers


225
Tiragolumab Heavy chain


226
Tiragolumab light chain


227
Relatlimab Heavy chain


228
Relatlimab Light chain


229
Second Chain (Heavy chain of anti-CTLA-4 antibody +



linker + human IL-21) with knob mutation


230
αCTLA-4 + linker + IL21RαWT


231-232
αCTLA-4 + linker + IL21RαMutein


233
Second Chain (Heavy chain of anti-TIGIT antibody +



linker + human IL-21) with knob mutation


234
αTIGIT + linker + IL21RαWT


235-236
αTIGIT + linker + IL21RαMutein


237
Second Chain (Heavy chain of anti-LAG-3 antibody +



linker + human IL-21) with knob mutation


238
αLAG-3 + linker + IL21RαWT


239-240
αLAG-3 + linker + IL21RαMutein




















SEQ ID NO
Sequence







1
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


(Nivolumab
LEWVAVIWYDGSKRYY


heavy chain)
ADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWG



QGTLVTVSSASTKGPS



VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF



PAVLQSSGLYSLSS



VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE



FLGGPSVFLFPPKP



KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP



REEQFNSTYRVVSVLT



VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP



PSQEEMTKNQVSLTC



LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD



KSRWQEGNVFSCSV



MHEALHNHYTQKSLSLSLGK





2
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL


(Nivolumab light
IYDASNRATGIPA


chain)
RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK



RTVAAPSVFIFPP



SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT



EQDSKDSTYSLSSTLT



LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC





3
QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQ


(pembrolizumab
GLEWMGGINPSNGGTNF


heavy chain)
NEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDM



GFDYWGQGTTVTVSS



ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL



TSGVHTFPAVLQSS



GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP



CPPCPAPEFLGGPSV



FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH



NAKTKPREEQFNSTY



RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE



PQVYTLPPSQEEMTK



NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL



YSRLTVDKSRWQEG



NVFSCSVMHEALHNHYTQKSLSLSLGK





4
EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQ


(pembrolizumab
APRLLIYLASYLES


light chain)
GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTK



VEIKRTVAAPSVF



IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE



SVTEQDSKDSTYSLS



STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC





5
EVQLLESGGV LVQPGGSLRL SCAASGFTFS NFGMTWVRQA


(cemiplimab
PGKGLEWVSG ISGGGRDTYF ADSVKGRFTI SRDNSKNTLY


heavy chain)
LQMNSLKGED TAVYYCVKWG NIYFDYWGQG TLVTVSSAST



KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS



GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC



NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF



PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE



VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV



SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV



SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS



FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL



SLGK





6
DIQMTQSPSS LSASVGDSIT ITCRASLSIN TFLNWYQQKP


(cemiplimab light
GKAPNLLIYA ASSLHGGVPS


chain)
RFSGSGSGTD FTLTIRTLQP EDFATYYCQQ SSNTPFTFGP



GTVVDFRRTV AAPSVFIFPP



SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ



ESVTEQDSKD STYSLSSTLT



LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC





7
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGL


(atezolizumab
EWVAWISPYGGSTYY


heavy chain)
ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGG



FDYWGQGTLVTVSSAS



TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS



GVHTFPAVLQSSGL



YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH



TCPPCPAPELLGGPS



VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV



HNAKTKPREEQYAST



YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR



EPQVYTLPPSREEMT



KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF



LYSKLTVDKSRWQQ



GNVFSCSVMHEALHNHYTQKSLSLSPGK





8
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPK


(atezolizumab
LLIYSASFLYSGVPS


light chain)
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK



RTVAAPSVFIFPP



SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT



EQDSKDSTYSLSSTLT



LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC





9
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGL


(dostarlimab
EWVSTISGGGSYTYY


heavy chain)
QDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYYAMD



YWGQGTTVTVSSASTK



GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV



HTFPAVLQSSGLYS



LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP



APEFLGGPSVFLFP



PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK



TKPREEQFNSTYRVVS



VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY



TLPPSQEEMTKNQVS



LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL



TVDKSRWQEGNVFS



CSVMHEALHNHYTQKSLSLSLGK





10
DIQLTQSPSFLSAYVGDRVTITCKASQDVGTAVAWYQQKPGKAPKL


(dostarlimab light
LIYWASTLHTGVPS


chain)
RFSGSGSGTEFTLTISSLQPEDFATYYCQHYSSYPWTFGQGTKLEIK



RTVAAPSVFIFPP



SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT



EQDSKDSTYSLSSTLT



LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC





11
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKG


(durvalumab
LEWVANIKQDGSEKYY


heavy chain)
VDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFG



ELAFDYWGQGTLVTVS



SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA



LTSGVHTFPAVLQS



SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK



THTCPPCPAPEFEG



GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG



VEVHNAKTKPREEQY



NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKG



QPREPQVYTLPP SRE



EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD



GSFFLYSKLTVDKSR



WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK





12
EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPR


(durvalumab light
LLIYDASSRATGIP


chain)
DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVE



IKRTVAAPSVFIFP



PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV



TEQDSKDSTYSLSSTL



TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC





13
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGL


(avelumab heavy
EWVSSIYPSGGITFY


chain)
ADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVT



TVDYWGQGTLVTVSS



ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL



TSGVHTFPAVLQSS



GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT



HTCPPCPAPELLGG



PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV



EVHNAKTKPREEQYN



STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ



PREPQVYTLPPSRDE



LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSKLTVDKSRW



QQGNVFSCSVMHEALHNHYTQKSLSLSPGK





14
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAP


(avelumab light
KLMIYDVSNRPSGV


chain)
SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTK



VTVLGQPKANPTVT



LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETT



KPSKQSNNKYAASS



YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS





15
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


IL-21RαWT
CSLHRSAHNATHATY



TCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFN



VTVTFSGQYNISWR



SDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVS



LLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEE



LKE





16
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


Human IgG1 Fc
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(100 Pro-330
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


Lys)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGK





17
GGGGSGGGGSGGGGS


G4S linker






18
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDKYEELKDEATS


Q35K
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





19
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDRYEELKDEATS


Q35R
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





20
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDYYEELKDEATS


Q35Y
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





21
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQCEELKDEATS


Y36C
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





22
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQEEELKDEATS


Y36E
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





23
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQGEELKDEATS


Y36G
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





24
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQHEELKDEATS


Y36H
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





25
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQIEELKDEATSC


Y36I
SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG


(IL-21Rα mutein)
SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY



ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA



GPMPGSSYQGTWSEWSDPVIFQTQSEELKE





26
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQKEELKDEATS


Y36K
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





27
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQMEELKDEATS


Y36M
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





28
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQNEELKDEATS


Y36N
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





29
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQPEELKDEATSC


Y36P
SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG


(IL-21Rα mutein)
SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY



ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA



GPMPGSSYQGTWSEWSDPVIFQTQSEELKE





30
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQQEELKDEATS


Y36Q
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





31
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQREELKDEATS


Y36R
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





32
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQSEELKDEATSC


Y36S
SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG


(IL-21Rα mutein)
SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY



ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA



GPMPGSSYQGTWSEWSDPVIFQTQSEELKE





33
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQTEELKDEATS


Y36T
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





34
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQVEELKDEATS


Y36V
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





35
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYECLKDEATS


E38C
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





36
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEKLKDEATS


E38K
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





37
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYERLKDEATS


E38R
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





38
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEYLKDEATS


E38Y
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





39
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEECKDEATS


L39C
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





40
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEEKDEATS


L39E
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





41
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEFKDEATS


L39F
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





42
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEHKDEATS


L39H
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





43
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEKKDEATS


L39K
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





44
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEERKDEATS


L39R
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





45
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEWKDEATS


L39W
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





46
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEYKDEATS


L39Y
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





47
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70C
CSLHRSAHNATHATYTCHMDVFHFCADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





48
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70D
CSLHRSAHNATHATYTCHMDVFHFDADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





49
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70F
CSLHRSAHNATHATYTCHMDVFHFFADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





50
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70G
CSLHRSAHNATHATYTCHMDVFHFGADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





51
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70H
CSLHRSAHNATHATYTCHMDVFHFHADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





52
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70K
CSLHRSAHNATHATYTCHMDVFHFKADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





53
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70L
CSLHRSAHNATHATYTCHMDVFHFLADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





54
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70N
CSLHRSAHNATHATYTCHMDVFHFNADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





55
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70Q
CSLHRSAHNATHATYTCHMDVFHFQADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





56
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70R
CSLHRSAHNATHATYTCHMDVFHFRADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





57
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70S
CSLHRSAHNATHATYTCHMDVFHFSADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





58
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70T
CSLHRSAHNATHATYTCHMDVFHFTADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





59
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70V
CSLHRSAHNATHATYTCHMDVFHFVADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





60
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70W
CSLHRSAHNATHATYTCHMDVFHFWADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





61
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M70Y
CSLHRSAHNATHATYTCHMDVFHFYADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





62
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


A71E
CSLHRSAHNATHATYTCHMDVFHFMEDDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





63
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


A71F
CSLHRSAHNATHATYTCHMDVFHFMFDDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





64
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


A71I
CSLHRSAHNATHATYTCHMDVFHFMIDDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





65
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


A71L
CSLHRSAHNATHATYTCHMDVFHFMLDDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





66
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


A71Q
CSLHRSAHNATHATYTCHMDVFHFMQDDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





67
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


A71R
CSLHRSAHNATHATYTCHMDVFHFMRDDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





68
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


A71W
CSLHRSAHNATHATYTCHMDVFHFMWDDIFSVNITDQSGNYSQE


(IL-21Rα mutein)
CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





69
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


A71Y
CSLHRSAHNATHATYTCHMDVFHFMYDDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





70
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72A
CSLHRSAHNATHATYTCHMDVFHFMAADIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





71
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72C
CSLHRSAHNATHATYTCHMDVFHFMACDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





72
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72E
CSLHRSAHNATHATYTCHMDVFHFMAEDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





73
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72F
CSLHRSAHNATHATYTCHMDVFHFMAFDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





74
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72G
CSLHRSAHNATHATYTCHMDVFHFMAGDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





75
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72H
CSLHRSAHNATHATYTCHMDVFHFMAHDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





76
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72I
CSLHRSAHNATHATYTCHMDVFHFMAIDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





77
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72K
CSLHRSAHNATHATYTCHMDVFHFMAKDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





78
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72L
CSLHRSAHNATHATYTCHMDVFHFMALDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





79
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72M
CSLHRSAHNATHATYTCHMDVFHFMAMDIFSVNITDQSGNYSQE


(IL-21Rα mutein)
CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





80
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72Q
CSLHRSAHNATHATYTCHMDVFHFMAQDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





81
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72R
CSLHRSAHNATHATYTCHMDVFHFMARDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





82
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72W
CSLHRSAHNATHATYTCHMDVFHFMAWDIFSVNITDQSGNYSQE


(IL-21Rα mutein)
CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





83
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D72Y
CSLHRSAHNATHATYTCHMDVFHFMAYDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





84
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D73C
CSLHRSAHNATHATYTCHMDVFHFMADCIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





85
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D73A
CSLHRSAHNATHATYTCHMDVFHFMADAIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





86
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D73E
CSLHRSAHNATHATYTCHMDVFHFMADEIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





87
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D73H
CSLHRSAHNATHATYTCHMDVFHFMADHIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





88
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D73K
CSLHRSAHNATHATYTCHMDVFHFMADKIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





89
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D73R
CSLHRSAHNATHATYTCHMDVFHFMADRIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





90
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D73W
CSLHRSAHNATHATYTCHMDVFHFMADWIFSVNITDQSGNYSQE


(IL-21Rα mutein)
CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





91
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


D73Y
CSLHRSAHNATHATYTCHMDVFHFMADYIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





92
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


I74H
CSLHRSAHNATHATYTCHMDVFHFMADDHFSVNITDQSGNYSQE


(IL-21Rα mutein)
CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





93
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


I74K
CSLHRSAHNATHATYTCHMDVFHFMADDKFSVNITDQSGNYSQE


(IL-21Rα mutein)
CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





94
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


I74R
CSLHRSAHNATHATYTCHMDVFHFMADDRFSVNITDQSGNYSQE


(IL-21Rα mutein)
CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





95
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


I74W
CSLHRSAHNATHATYTCHMDVFHFMADDWFSVNITDQSGNYSQE


(IL-21Rα mutein)
CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





96
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


L94F
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFFLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





97
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


L94K
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFKLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





98
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


L94Q
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFQLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





99
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


L94R
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFRLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





100
QDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAF


Human IL-21
SCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTC



PSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS





101
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


second chain
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(Heavy chain of
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


anti-PD-1
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


antibody +
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF


linker +
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV


human IL-21 with
DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK


a knob mutation
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCL



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW



SAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHR



LTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS





102
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL


Anti-PD-1
IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNW


antibody, Light
PRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP


chain
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD



YEKHKVYACEVTHQGLSSPVTKSFNRGEC





103
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Anti-PD-1
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


antibody, Heavy
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


chain with a hole
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


mutation
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG





104
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36C
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQCEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





105
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36E
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQEEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





106
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36G
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQGEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





107
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36H
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQHEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





108
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36I
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQIEELKDEATSC



SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG



SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY



ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA



GPMPGSSYQGTWSEWSDPVIFQTQSEELKE





109
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36K
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQKEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





110
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36M
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQMEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





111
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36N
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQNEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





112
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36P
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQPEELKDEATSC



SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG



SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY



ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA



GPMPGSSYQGTWSEWSDPVIFQTQSEELKE





113
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36Q
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQQEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





114
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36R
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQREELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





115
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36S
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQSEELKDEATSC



SLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECG



SFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQY



ELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRA



GPMPGSSYQGTWSEWSDPVIFQTQSEELKE





116
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36T
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQTEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





117
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


Y36V
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQVEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





118
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


E38C
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYECLKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





119
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


E38Y
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEYLKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





120
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L39C
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEECKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





121
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L39E
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEEKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





122
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L39H
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEHKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





123
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L39K
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEKKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





124
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L39R
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEERKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





125
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L39W
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEWKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





126
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L39Y
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEYKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





127
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70F
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFFADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





128
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70H
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFHADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





129
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70N
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFNADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





130
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70Q
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFQADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





131
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70S
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFSADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





132
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70T
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFTADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





133
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70V
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFVADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





134
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70W
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFWADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





135
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70Y
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFYADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





136
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


A71E
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMEDDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





137
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


A71F
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMFDDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





138
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


A71I
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMIDDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





139
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


A71L
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMLDDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





140
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


A71Q
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMQDDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





141
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


A71R
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMRDDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





142
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


A71W
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMWDDIFSVNITDQSGNYSQE



CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





143
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


A71Y
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMYDDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





144
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


I74R
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDRFSVNITDQSGNYSQE



CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





145
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


I74W
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDWFSVNITDQSGNYSQE



CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





146
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L94F
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFFLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





147
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L94K
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFKLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





148
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L94Q
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFQLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





149
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L94R
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFRLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





150
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


L94Y
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFYLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





151 (Ipilimumab
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG


heavy chain)
LEWVTFISYDGNNKYY



ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPF



DYWGQGTLVTVSSAS



TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS



GVHTFPAVLQSSGL



YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT



CPPCPAPELLGGPS



VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV



HNAKTKPREEQYNST



YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR



EPQVYTLPPSRDELT



KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF



LYSKLTVDKSRWQQ



GNVFSCSVMHEALHNHYTQKSLSLSPGK





152 (Ipilimumab
EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPR


light chain)
LLIYGAFSRATGIP



DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEI



KRTVAAPSVFIFP



PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV



TEQDSKDSTYSLSSTL



TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC





153
QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA


(tremelimumab
PGKGLEWVAV IWYDGSNKYY


heavy chain)
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP



RGATLYYYYY GMDVWGQGTT



VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE



PVTVSWNSGA LTSGVHTFPA



VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD



KTVERKCCVE CPPCPAPPVA



GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN



WYVDGVEVHN AKTKPREEQF



NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK GLPAPIEKTI



SKTKGQPREP QVYTLPPSRE



EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP



MLDSDGSFFL YSKLTVDKSR



WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K





154
DIQMTQSPSS LSASVGDRVT ITCRASQSIN SYLDWYQQKP


(tremelimumab
GKAPKLLIYA ASSLQSGVPS


light chain)
RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYSTPFTFGP



GTKVEIKRTV AAPSVFIFPP



SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ



ESVTEQDSKD STYSLSSTLT



LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC





155
CPDLVCYTDALQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


Y10A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





156
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQAEELKDEATS


Y36A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





157
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEALKDEATS


E38A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





158
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEEAKDEATS


L39A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





159
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


F67A
CSLHRSAHNATHATYTCHMDVAHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





160
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


H68A
CSLHRSAHNATHATYTCHMDVFAFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





161
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


I74A
CSLHRSAHNATHATYTCHMDVFHFMADDAFSVNITDQSGNYSQE


(IL-21Rα mutein)
CGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKL



QYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQV



RAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





162
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


L94A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFALAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





163
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


P126A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDAAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





164
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


Y129A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFAMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





165
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


M130A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYALKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





166
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


K134A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGALQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





167
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


S189A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGASYQGTWSEWSDPVIFQTQSEELKE





168
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


S190A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSAYQGTWSEWSDPVIFQTQSEELKE





169
CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS


Y191A
CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC


(IL-21Rα mutein)
GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSAQGTWSEWSDPVIFQTQSEELKE





170
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


Y10A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDALQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





171
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


Y36A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQAEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





172
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


E38A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEALKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





173
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


L39A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEEAKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





174
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


F67A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVAHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





175
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


H68A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFAFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





176
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


I74A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDAFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





177
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


L94A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21 Ra mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFALAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





178
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


P126A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDAAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





179
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


Y129A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFAMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





180
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


M130A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYALKGKLQYELQYRNRGDPWAVSPRRKLISVDSR



SVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQ



SEELKE





181
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


K134A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGALQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQT



QSEELKE





182
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


S189A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGASYQGTWSEWSDPVIFQT



QSEELKE





183
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


S190A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSAYQGTWSEWSDPVIFQT



QSEELKE





184
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


Y191A
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(Fc-linker-IL-
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP


21Rα mutein)
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGKGGGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPS



TLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFM



ADDIFSVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNI



SWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS



RSVSLLPLEFRKDSSYELQVRAGPMPGSSAQGTWSEWSDPVIFQT



QSEELKE





185
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV


IgG1 Fc moiety
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


(WT)
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP



PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPGK





186
ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD


IgG2 Fc moiety
VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVV


(WT)
HQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS



REEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLD



SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL



SLSPGK





187
ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP


IgG3 Fc moiety
EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV


(WT)
VVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVL



TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTL



PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPM



LDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL



SPGK





188
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD


IgG4 Fc moiety
VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL


(WT)
HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS



QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD



SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS



LGK





189
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL


IGHG1
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK


(Immunoglobulin
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT


heavy constant
PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST


gamma 1) with
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR


‘LALA’(L234A/
EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN


L235A) mutation
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH



YTQKSLSLSPGK





190
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL


IGHG4
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT


(Immunoglobulin
KVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEV


heavy constant
TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV


gamma 4) with
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ


SPLE‘(S228P/L
VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK


235E) mutation
TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT



QKSLSLSLGK





191
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


αPD-1-linker-
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


IL21RαWT
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST



AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV



VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGS



CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATS



CSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQEC



GSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQ



YELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVR



AGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





192
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG


Y36A + D72E
KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN


(αPD-
SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP


1 + linker + IL21Rα
CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES



KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV



DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV



LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV



YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY



KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH



NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV



ICILEMWNLHPSTLTLTWQDQAEELKDEATSCSLHRSAHNATH



ATYTCHMDVFHFMAEDIFSVNITDQSGNYSQECGSFLLAESIK



PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR



NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP



MPGSSYQGTWSEWSDPVIFQTQSEELKE





193
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG


Y36A + L94R
KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN


(αPD-
SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP


1 + linker + IL21Rα
CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES



KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV



DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV



LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV



YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY



KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH



NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV



ICILEMWNLHPSTLTLTWQDQAEELKDEATSCSLHRSAHNATH



ATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFRLAESIK



PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR



NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP



MPGSSYQGTWSEWSDPVIFQTQSEELKE





194
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG


E38A + D72E
KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN


(αPD-
SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP


1 + linker + IL21Rα
CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES



KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV



DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV



LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV



YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY



KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH



NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV



ICILEMWNLHPSTLTLTWQDQYEALKDEATSCSLHRSAHNATH



ATYTCHMDVFHFMAEDIFSVNITDQSGNYSQECGSFLLAESIK



PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR



NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP



MPGSSYQGTWSEWSDPVIFQTQSEELKE





195
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG


E38A + L94K
KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN


(αPD-
SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP


1 + linker + IL21Rα
CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES



KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV



DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV



LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV



YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY



KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH



NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV



ICILEMWNLHPSTLTLTWQDQYEALKDEATSCSLHRSAHNATH



ATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFKLAESIK



PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR



NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP



MPGSSYQGTWSEWSDPVIFQTQSEELKE





196
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG


E38A + L94R
KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN


(αPD-
SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP


1 + linker + IL21Rα
CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES



KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV



DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV



LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV



YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY



KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH



NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV



ICILEMWNLHPSTLTLTWQDQYEALKDEATSCSLHRSAHNATH



ATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFRLAESIK



PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR



NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP



MPGSSYQGTWSEWSDPVIFQTQSEELKE





197
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG


E38R + D72R
KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN


(αPD-
SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP


1 + linker + IL21Rα
CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES



KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV



DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV



LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV



YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY



KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH



NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV



ICILEMWNLHPSTLTLTWQDQYERLKDEATSCSLHRSAHNATH



ATYTCHMDVFHFMARDIFSVNITDQSGNYSQECGSFLLAESIK



PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR



NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP



MPGSSYQGTWSEWSDPVIFQTQSEELKE





198
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG


D72E + L94K
KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN


(αPD-
SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP


1 + linker + IL21Rα
CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES



KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV



DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV



LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV



YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY



KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH



NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV



ICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATH



ATYTCHMDVFHFMAEDIFSVNITDQSGNYSQECGSFKLAESIK



PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR



NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP



MPGSSYQGTWSEWSDPVIFQTQSEELKE





199
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPG


D72E + L94R
KGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMN


(αPD-
SLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP


1 + linker + IL21Rα
CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES



KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV



DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV



LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV



YTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY



KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALH



NHYTQKSLSLSLGGGGGSGGGGSGGGGSCPDLVCYTDYLQTV



ICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATH



ATYTCHMDVFHFMAEDIFSVNITDQSGNYSQECGSFRLAESIK



PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYR



NRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGP



MPGSSYQGTWSEWSDPVIFQTQSEELKE





200
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


E38R
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYERLKDE



ATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





201
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70C
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE



ATSCSLHRSAHNATHATYTCHMDVFHFCADDIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





202
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70D
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE



ATSCSLHRSAHNATHATYTCHMDVFHFDADDIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





203
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70G
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE



ATSCSLHRSAHNATHATYTCHMDVFHFGADDIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





204
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


M70R
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE



ATSCSLHRSAHNATHATYTCHMDVFHFRADDIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





205
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


D72A
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE



ATSCSLHRSAHNATHATYTCHMDVFHFMAADIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





206
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


D72E
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE



ATSCSLHRSAHNATHATYTCHMDVFHFMAEDIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





207
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


D72Q
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE



ATSCSLHRSAHNATHATYTCHMDVFHFMAQDIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





208
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


D72R
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE



ATSCSLHRSAHNATHATYTCHMDVFHFMARDIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





209
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKG


D73A
LEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAE


(αPD-
DTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST


1 + linker + IL21Rα
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV


Mutein)
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF



LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV



DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK



VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCA



VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDK



SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGG



GGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDE



ATSCSLHRSAHNATHATYTCHMDVFHFMADAIFSVNITDQSGNYS



QECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKG



KLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYEL



QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





210
CCGAAATCATGTGACAAAACTCATACTTGTCCTCCATGCCCA


(Polynucleotide
GCCCCAGAATTGCTGGGGGGACCATCTGTGTTCCTTTTCCCC


encoding IgG1
CCTAAGCCAAAAGACACTCTGATGATCAGTCGCACTCCTGA


Fc-IL21Rα
AGTGACCTGCGTCGTGGTAGACGTCTCTCACGAAGATCCCG


(wildtype))
AGGTCAAATTTAACTGGTATGTGGATGGCGTGGAAGTTCATA



ACGCAAAAACCAAACCCCGCGAAGAACAATATAATAGCACA



TACCGTGTTGTTAGCGTTTTGACAGTCCTTCACCAGGATTGG



CTCAACGGAAAAGAGTACAAGTGCAAGGTGTCCAATAAAG



CATTGCCCGCCCCTATAGAGAAGACTATTAGCAAGGCCAAA



GGTCAGCCCCGGGAGCCTCAGGTGTATACATTGCCTCCCAG



CCGCGATGAACTCACTAAAAACCAAGTCAGCCTCACATGTC



TGGTTAAAGGTTTTTACCCCAGCGATATCGCAGTCGAGTGGG



AATCTAATGGGCAGCCTGAAAATAACTATAAGACAACCCCA



CCAGTGTTGGATAGCGATGGCAGCTTTTTTCTTTACTCTAAG



TTGACTGTTGACAAGAGCAGGTGGCAACAAGGCAACGTGT



TTAGCTGCAGTGTCATGCACGAAGCACTCCACAATCATTACA



CCCAGAAGAGTCTGAGCTTGTCACCTGGAAAGGGTGGAGG



CGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCATGTC



CTGACCTGGTGTGCTACACCGACTACCTGCAGACCGTGATC



TGCATCCTGGAGATGTGGAACCTGCATCCTTCTACCCTGACA



CTGACCTGGCAGGACCAGTACGAGGAACTGAAGGACGAGG



CCACCTCCTGCTCCCTGCACAGATCTGCTCACAACGCCACC



CACGCTACCTACACCTGTCACATGGACGTGTTCCACTTCATG



GCCGACGACATCTTTTCTGTGAACATCACCGATCAGTCTGGC



AACTACTCCCAAGAGTGCGGCTCTTTCCTGCTGGCCGAGTC



CATCAAGCCTGCTCCTCCTTTCAACGTGACCGTGACCTTCTC



CGGCCAGTACAACATCTCTTGGCGGTCCGACTACGAGGACC



CCGCCTTCTACATGCTGAAGGGCAAGCTGCAGTACGAGCTG



CAGTACCGGAACAGAGGCGACCCTTGGGCCGTGTCCCCTAG



AAGAAAGCTGATCTCCGTGGACTCCAGATCCGTGTCTCTGC



TGCCTCTGGAATTCCGGAAGGACTCTAGCTACGAACTGCAA



GTGCGGGCTGGCCCTATGCCTGGCTCCTCCTACCAGGGAAC



ATGGTCCGAGTGGAGCGATCCTGTGATCTTCCAGACCCAGT



CCGAAGAGCTGAAAGAG





211
CAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGCAG


(Polynucleotide
CCAGGCAGGTCCCTGCGGCTGGACTGTAAGGCCTCCGGCA


encoding αPD-1
TCACCTTTTCTAACTCCGGAATGCATTGGGTGAGGCAGGCT


IL21RαWT)
CCAGGCAAGGGCCTGGAGTGGGTGGCTGTGATCTGGTACG



ACGGCAGCAAGCGGTACTATGCCGATTCTGTGAAGGGCAG



ATTCACAATCTCTCGCGACAACTCCAAGAATACCCTGTTTC



TGCAGATGAACTCTCTGAGGGCCGAGGATACAGCCGTGTA



CTATTGCGCTACCAATGACGATTACTGGGGCCAGGGCACAC



TGGTGACCGTGTCCAGCGCCAGCACAAAGGGACCATCCGT



GTTCCCACTGGCTCCATGCAGCCGGTCTACATCCGAGAGCA



CCGCCGCTCTGGGATGTCTGGTGAAGGATTATTTCCCTGAG



CCAGTGACCGTGAGCTGGAACTCCGGCGCCCTGACATCTG



GCGTGCACACCTTTCCTGCTGTGCTGCAGTCTTCCGGCCTG



TACTCCCTGAGCTCTGTGGTGACAGTGCCCTCCAGCTCTCT



GGGCACCAAGACATATACCTGCAACGTGGACCATAAGCCTT



CCAATACCAAGGTGGATAAGAGAGTGGAGAGCAAGTACGG



ACCACCTTGCCCACCATGTCCAGCTCCTGAGTTTCTGGGAG



GACCATCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACC



CTGATGATCAGCCGCACACCTGAGGTGACCTGCGTGGTGG



TGGACGTGTCTCAGGAGGACCCCGAGGTGCAGTTCAACTG



GTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACCAAG



CCTAGAGAGGAGCAGTTTAACTCCACATACCGCGTGGTGA



GCGTGCTGACCGTGCTGCATCAGGACTGGCTGAACGGCAA



GGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCATCCA



GCATCGAGAAGACAATCAGCAAGGCCAAGGGCCAGCCTAG



GGAGCCACAGGTGTACACCCTGCCCCCTTCTCAGGAGGAG



ATGACAAAGAACCAGGTGTCCCTGTCCTGTGCCGTGAAGG



GCTTCTATCCAAGCGACATCGCTGTGGAGTGGGAGTCTAAT



GGCCAGCCCGAGAACAATTACAAGACCACACCACCCGTGC



TGGACTCCGATGGCAGCTTCTTTCTGGTCTCCAGGCTGACA



GTGGATAAGAGCCGGTGGCAGGAGGGCAACGTGTTTTCTT



GTTCCGTGATGCACGAGGCTCTGCACAATCATTACACCCAG



AAGAGCCTGTCTCTGTCCCTGGGCGGTGGCGGTGGCTCTG



GCGGAGGTGGCTCAGGTGGCGGCGGATCCTGTCCTGATCT



CGTGTGCTATACCGACTACCTCCAGACCGTTATTTGTATCCT



TGAGATGTGGAATTTGCACCCATCAACACTGACTCTGACTT



GGCAGGATCAATACGAGGAGCTGAAAGACGAGGCCACATC



CTGCTCCTTGCATCGATCAGCACACAACGCCACTCATGCAA



CATACACTTGCCATATGGATGTGTTCCACTTCATGGCAGATG



ATATTTTTTCAGTTAACATTACAGATCAATCCGGCAACTATT



CACAGGAATGTGGCTCTTTTCTTCTGGCAGAATCAATAAAG



CCCGCACCTCCTTTCAACGTGACTGTCACCTTCTCAGGACA



ATATAATATCAGCTGGCGATCTGACTATGAGGACCCTGCCTT



TTACATGCTGAAAGGCAAGCTCCAATACGAACTTCAATATC



GTAATAGGGGGGACCCATGGGCCGTCAGTCCTCGACGGAA



GCTGATATCCGTGGACTCTAGAAGTGTCTCTCTCTTGCCCCT



CGAATTTAGGAAAGACTCATCCTACGAGCTTCAAGTTCGGG



CAGGTCCCATGCCCGGCTCAAGCTATCAGGGGACATGGAG



CGAGTGGTCCGACCCAGTAATTTTCCAAACCCAAAGCGAG



GAATTGAAAGAG





212
GGGGS


(GGGGS)1



Flexible Linker






213
GGGGSGGGGS


(GGGGS)2



Flexible Linker






214
GGGGSGGGGSGGGGS


(GGGGS)3



Flexible Linker






215
GGGGSGGGGSGGGGSGGGGS


(GGGGS)4



Flexible Linker






216
GGGGGG


(Gly)6



Flexible Linker






217
GGGGGGGG


(Gly)8



Flexible Linker






218
EAAAK


(EAAAK)1



Rigid Linker






219
EAAAKEAAAK


(EAAAK)2



Rigid Linker






220
EAAAKEAAAKEAAAK


(EAAAK)3



Rigid Linker






221
AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAA


A(EAAAK)4ALE
KEAAAKA


A(EAAAK)4A



Rigid Linker






222
PAPAP


PAPAP



Rigid Linker






223
AEAAAKEAAAKA


AEAAAKEAAAKA



Rigid Linker






224
(AP)n, (n = 5-15)


(Ala-Pro)n



(10-33 aa)



Rigid Linker






225
EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG


Tiragolumab
LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP


Heavy chain
EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL



APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL



QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC



DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS



HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ



DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE



MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG



SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK





226
DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQK


Tiragolumab light
PGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY


chain
YCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV



CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS



TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC





227
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG


Relatlimab Heavy
LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT


chain
AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS



RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG



LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP



PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ



FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK



EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV



SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR



LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK





228
EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLI


Relatlimab Light
YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP


chain
LTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR



EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE



KHKVYACEVTHQGLSSPVTKSFNRGEC





229

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG



Second Chain

LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE



(Heavy chain of

DTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSK



anti-CTLA-4

STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL



antibody + linker +

YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT



human IL-21)

CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE



with knob

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN



mutation

GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN





QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL





YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGG





SGGGGSGGGGSQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPE





DVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTN





AGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTH





GSEDS






230

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG



αCTLA-4 +

LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE



linker + IL21Rα

DTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSK



WT

STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL





YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT





CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE





VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN





GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN





QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV





SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS





GGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ





YEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNIT





DQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDP





AFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEF





RKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE






231

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG



M70D

LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE



(αCTLA-4 +

DTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSK



linker + IL21Rα

STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL



mutein)

YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT





CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE





VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN





GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN





QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV





SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS





GGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ





YEELKDEATSCSLHRSAHNATHATYTCHMDVFHFDADDIFSVNIT





DQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDP





AFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEF





RKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE






232

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG



D72A

LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE



(αCTLA-4 +

DTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSK



linker + IL21Rα

STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL



mutein)

YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT





CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE





VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN





GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN





QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV





SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS





GGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQ





YEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMAADIFSVNIT





DQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDP





AFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEF





RKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE






233

EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG



Second Chain

LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP



(Heavy chain of

EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL



anti-TIGIT

APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL



antibody +

QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC



linker + human

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS



IL-21) with knob

HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ



mutation

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE





MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD





GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG





GGGGSGGGGSGGGGSQDRHMIRMRQLIDIVDQLKNYVNDLVPEF





LPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRK





PPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHL





SSRTHGSEDS






234

EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG



αTIGIT +

LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP



linker + IL21Rα

EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL



WT

APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL





QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC





DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS





HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ





DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE





MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG





SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGG





GGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLT





WQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIF





SVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSD





YEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLL





PLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELK





E






235
EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG


M70D
LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP


(αTIGIT +
EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL


linker + IL21Rα
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC



DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS



HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ



DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE



MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG



SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGG



GGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLT



WQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFDADDIF



SVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSD



YEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLL



PLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELK



E





236
EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRG


D72A
LEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTP


(αTIGIT +
EDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPL


linker + IL21Rα
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL


Mutein)
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC



DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS



HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ



DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE



MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG



SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGG



GGGSGGGGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLT



WQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMAADIF



SVNITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSD



YEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLL



PLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELK



E





237

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG



Second Chain

LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT



(Heavy chain of

AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS



anti-LAG-3

RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG



antibody + linker +

LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP



human IL-21)

PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ



with knob

FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK



mutation

EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV





SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR





LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGG





GGSGGGGSQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVE





TNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGR





RQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSE





DS






238
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG


αLAG-3 +
LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT


linker + IL21Rα
AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS


WT
RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG



LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP



PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ



FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK



EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV



SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSR



LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGG



GGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE



ELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQ



SGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFY



MLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKD



SSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE





239

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG



M70D

LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT



(αLAG-3 +

AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS



inker + IL21Rα

RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG



Mutein)

LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP





PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ





FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK





EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV





SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSR





LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGG





GGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE





ELKDEATSCSLHRSAHNATHATYTCHMDVFHFDADDIFSVNITDQS





GNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFY





MLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKD





SSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE






240
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKG


D72A
LEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADT


(αLAG-3 +
AVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCS


linker + IL21Rα
RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG


Mutein)
LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP



PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ



FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK



EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV



SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSR



LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGG



GGSGGGGSCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYE



ELKDEATSCSLHRSAHNATHATYTCHMDVFHFMAADIFSVNITDQ



SGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYEDPAFY



MLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKD



SSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKE








Claims
  • 1. An immunocytokine, comprising: A. an antigen binding protein (ABP) specific to a target protein, optionally wherein the target protein is an immune checkpoint molecule;B. an IL-21 domain; andC. an IL-21Rα mutein,wherein the IL-21Rα mutein has a reduced binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.
  • 2. (canceled)
  • 3. The immunocytokine of claim 1, wherein the target protein is PD-1, PD-L1, TIGIT, LAG-3, CTLA-4, TIM-3, CD39, CD38, CD73, CD36, CD25, CD47, CD24, CD20, SIPRα, CD40, or CD20.
  • 4. (canceled)
  • 5. The immunocytokine of claim 1, wherein the ABP comprises Fc fragment, optionally selected from a human IgG1 Fc fragment, a human IgG2 Fc fragment, a human IgG3 Fc fragment, or a human IgG4 Fc fragment.
  • 6. (canceled)
  • 7. The immunocytokine of claim 1, wherein the ABP comprises Fc fragment, wherein the Fc fragment comprises a sequence selected from SEQ ID NOs: 16, 185-190.
  • 8. The immunocytokine of claim 1, wherein the ABP comprises an Fc fragment comprising two Fc moieties, and the IL-21Rα mutein is linked to the first of the two Fc moieties, and the IL-21 domain is linked to the second of the two Fc moieties.
  • 9. The immunocytokine of claim 8, wherein the IL-21 domain and the IL-21Rα mutein are respectively linked through a non-cleavable peptide linker or without a peptide linker.
  • 10. (canceled)
  • 11. The immunocytokine of claim 8, wherein the IL-21 domain and the IL-21Rα mutein are respectively linked through a non-cleavable peptide linker, wherein the non-cleavable peptide linker is G4S linker having the sequence of SEQ ID NO: 17 or a peptide linker having a sequence selected from SEQ ID NOs: 212-224.
  • 12. (canceled)
  • 13. The immunocytokine of claim 1, wherein the ABP comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences identical to an antibody selected from nivolumab, pembrolizumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, avelumab, ipilimumab, tiragolumab, relatlimab, and tremelimumab.
  • 14. The immunocytokine of claim 1, wherein the IL-21Rα mutein has 10 to 10,000-fold decrease in binding affinity to the IL-21 domain compared to a wild-type IL-21Rα.
  • 15. The immunocytokine of claim 1, wherein the IL-21Rα mutein has a sequence with at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15.
  • 16. (canceled)
  • 17. The immunocytokine of claim 1, wherein the IL-21Rα mutein comprises one to five amino acid substitutions compared to SEQ ID NO: 15, optionally wherein the one or more amino acid substitutions are at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 in SEQ ID NO: 15.
  • 18-19. (canceled)
  • 20. The immunocytokine of claim 1, wherein the IL-21Rα mutein comprises a sequence selected from SEQ ID NOs: 18-99 and 155-169.
  • 21. The immunocytokine of claim 1, comprising a first chain comprising from the N terminus to C terminus: A. a first Fc moiety or a first heavy chain of a human IgG1, IgG2, IgG3 or IgG4, wherein the first Fc moiety or the first heavy chain comprises a knob-and-hole mutation; andB. the IL-21Rα mutein.
  • 22. The immunocytokine of claim 21, wherein the IL-21Rα mutein comprises one or more amino acid mutations at one or more amino acid positions selected from Y10, Q35, Y36, E38, L39, F67, H68, M70, A71, D72, D73, I74, L94, P126, Y129, M130, K134, S189, S190, and Y191 in SEQ ID NO: 15.
  • 23. (canceled)
  • 24. The immunocytokine of claim 22, wherein the first chain comprises a sequence selected from SEQ ID NOs: 104-150, 192-209, 231-232, 235-236, and 239-240.
  • 25-26. (canceled)
  • 27. The immunocytokine of claim 1, comprising a second chain comprising a heavy chain of the ABP and the IL-21 domain, optionally wherein the second chain has the sequence of SEQ ID NO: 101, 229, 233 or 237.
  • 28. (canceled)
  • 29. The immunocytokine of claim 1, comprising a light chain having the sequence of SEQ ID NO: 102, 152, 226 or 228.
  • 30. One or more polynucleotides encoding the immunocytokine of claim 1.
  • 31-35. (canceled)
  • 36. A host cell comprising the one or more polynucleotides of claim 30.
  • 37-38. (canceled)
  • 39. A method of enhancing immune response or a method of treating cancer in a subject, comprising administration of the immunocytokine of claim 1 to the subject.
  • 40-42. (canceled)
  • 43. An IL-21Rα mutein having a reduced binding affinity to an IL-21 domain compared to a wild-type IL-21Rα.
  • 44-51. (canceled)
1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. Provisional Patent Application Nos. 63/250,911 filed on Sep. 30, 2021 and 63/351,298 filed on Jun. 10, 2022, the entire contents of which are incorporated by reference herein.

Provisional Applications (2)
Number Date Country
63250911 Sep 2021 US
63351298 Jun 2022 US