NZ791361B2 - Engineered anti-il-2 antibodies - Google Patents

Abstract

Described herein are engineered anti-IL-2 antibodies with modified amino acid sequences. The engineered antibodies would confer modified receptor binding specificity to an IL-2-anti-IL2 antibody complex, inhibiting the binding of IL-2 to CD25. The engineered anti-IL-2 antibodies would facilitate expansion of subsets of effector immune cells and decrease undesirable effects caused by IL-2. Thus, the engineered anti-IL-2 antibodies would be useful in treating disease such as cancer and infection.

Description

ENGINEERED ANTI-IL-2 ANTIBODIES CROSS-REFERENCE TO RELATED APPLICATIONS This Application claims benefit of priority of United States Provisional Application Nos. 62/977,292 filed February 16, 2020 and 63/139,315 filed January 20, 2021, which are all orated by nce herein in their entirety.

SEQUENCE LISTING STATEMENT The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, d on February 14, 2021, is named 94-PC-SEQ-l4FEB2l_ST25.txt and is 64.6 kilobytes in size.

FIELD OF THE ION The disclosure relates in general to the field of antibodies. In one ment, the t disclosure describes the making and uses of engineered anti-IL-2 dies that would confer modified receptor binding specificity to IL-2.

BACKGROUND Interleukin 2 (IL-2) is a 15.4 kDa type I cytokine with a four helix bundle structure. Since its discovery more than 30 years ago, the importance of IL—2 in regulation of the immune system has been illustrated many times. IL-2 is mostly produced and secreted by CD4+ T cells that are activated by an antigen. To a lesser extent, IL-2 is also produced by CD8+ T cells, natural killer (NK) cells, dendritic cells and mast cells.

IL-2 signaling has two opposite effects. IL-2 can enhance immune response by tion of effector cells and induce their proliferation. Alternatively, IL-2 can tune down immune response by activation and proliferation of CD4+ regulatory T (Treg) cells. To facilitate theses function, IL-2 mediates its effect by binding to two forms of IL-2 or: i) trimeric receptors made up of IL-2R0t (CD25), IL—2RB (CD122), and a common IL-2Rv (vc, CD132) chains, or ii) a dimeric receptor that consists of only the IL—2RB and IL-2Rv subunits. Both the dimeric and trimeric receptors are able to transmit IL-2 binding signaling via the STAT5 pathway. However, IL-2 binds the (1B7 receptor trimer a 100 fold tighter than the By receptor dimer. It has been demonstrated that the binding affinity of hIL- 2 to the (1B7 trimer is approximately 10pM, whereas the hIL-2 ty to the By dimer is lnM. id="p-6" id="p-6" id="p-6"

[0006] The difference in affinities to the dimeric and trimeric forms of the IL-2 receptor is one of the key isms in charge of keeping immunological homeostasis in vivo. Activation of the trimeric receptor is associated with FoxP3-mediated transcription in Tregs, which express on its ne more of the (xBy trimeric IL-2 receptor. In contrast, binding of IL-2 to the By dimer is associated with activation of NK cells and memory phenotype (MP) CD8+ cells, which express relatively high levels of the By dimer and very low levels the (xBy trimeric IL-2 receptor. Since in normal logical condition the l levels of IL-2 are relatively low, it seems the main function of IL-2 is to facilitate immune tolerance by acting as a Treg activating and proliferative . On the other hand, once the immune system is activated, IL-2 levels are rising and subsequently IL-2 is able to bind the By dimer and facilitate memory phenotype effector T cells (MP) CD8+ and NK cells activation and proliferation. id="p-7" id="p-7" id="p-7"

[0007] Since the early 1990’ s, high dose IL-2 therapy is used to treat melanoma and metastatic renal cell carcinoma with 10%-15% response rate. While efficacious, this approach has not been adopted to other cancers, since ILdependent adverse effects like the potentially lethal Vascular Leak Syndrome (VLS) excludes many patients from being considered for this therapy. The short half-life of administered IL-2 requires very frequent stration, resulting in ed spikes in the level of ating IL-2, thereby exacerbating adverse effects. Finally, since wild type IL-2 is not selective it could also enhance a non-desired activation of Treg cells.

It has been discovered that certain dies could bind to IL-2 and modulate g to the By dimeric or the (xBy trimeric IL-2 receptors. IL-2 complexed with these antibodies would have relatively longer half-life, and these IL-2 complexes would activate ic subsets of effector or immune cells. For example, the antibody S4B6-mouse IL-2 complex entially activates mouse effector cells in vivo, whereas the antibody JES6.l-mouse IL-2 complex preferentially activates mouse T regulatory cells in vivo. The mechanism of the modulation by the JES-6.1 antibody has been elucidated. It has been shown that the JES6.l-mIL-2 x binds CD25 but not CD 122 in vitro.

Increased IL-2 has been implicated to play a role in viral infection. SARS-CoV-2 is a positive stranded RNA virus of the coronaviridae family of respiratory viruses. The virus gains entry into the host by binding angiotensin converting enzyme 2 (ACE2) on lung and gastrointestinal tissues. The course of infection is characterized by a ~ 7-14 day incubation period followed by symptoms of a dry cough, fever, and shortness of breath. Up to 20% of symptomatic individuals develop severe symptoms and on average 3% of the cases are fatal due to pulmonary failure. Earlier studies with members of the coronavirus family demonstrated that coronavirus ions result in an increase in regulatory T lymphocytes, which likely contributes to delayed viral clearance. More recent studies on COVID-l9 patients showed patients in the ICU had higher levels of IL-2, IL-7, IL-10, GSCF, IP10, MCPl, MIPlA, and TNF-(x than non-ICU patients, suggesting a role of immunopathology in severe disease. Investigations into direct evidence of alterations in leukocyte homeostasis using the immunological characteristics of peripheral blood leukocytes from SARS-CoV2 infected patients indicate that in COVID-l9, similar to some chronic infections, damages to the function of CD4+ T cells promotes ive activation and possibly subsequent exhaustion of CD8+ T cells. These perturbations of T cell subsets may ally diminish host antiviral ty. Therapeutics that either slow viral growth or e the immune response to eliminate viral load while reducing some of the associated immune pathologies would therefore be of great benefit. id="p-10" id="p-10" id="p-10"

[0010] Immune responses to viruses consist of both the innate and acquired arms of the immune system. The innate system uses ike receptors (TLR) and retinoic acid inducible gene I (RIG-I) proteins to sense viral RNA/DNA and induce an initial response. This response includes the production of antiviral cytokines (such as interferon 0t), chemokines to bring the immune system to the site of the ion and mobilization of the macrophage/dendritic cell arm. Natural Killer (NK) cells (innate lymphoid) directly kill virus infected cells in the absence of MHC class-I expression.

This can occur even when the virus has interfered with the MHC class I presentation system.

In on, during an infection response NK cells produce interferon-y (IFN—y), y sing the expression of MHC Class I on cells and enhancing the ability of the acquired immune system to d. The acquired immune system consists of T cells (CD4 and CD8) and B cells.

CD4+ T cells recognize viral antigens in the context of MHC-II on antigen presenting cells to both amplify the immune response (through cytokines) and induce B cell class ing and subsequent production of anti-viral antibodies. Activation of CD4 cells, in particular Th1 cells, also releases IFN— y, thus enhancing the presentation of viral antigens. CD8+ T cells t direct lytic effects to virally infected cells which are presenting viral peptides in the context of MHC-I. The initial induction phase of the immune response typically takes 7 — 10 days to expand the T cell population and generates the cells required to clear the viruses.

IL-2 is a key mediator in the expansion and activation of T cells and NK cells. IL-2 is commonly thought to play a major role in the secondary s required for T cell activation. The expression of the dimeric (By) and trimeric (ocBy) IL-2 receptor complexes show lineage selectivity in that the ic receptor containing CD25 (the Qt t) is found highly expressed on regulatory T cells and a subset of activated short lived cytotoxic effector T cells, whereas the c receptor is found on naive T cells, memory T cells, and NK cells. Consequently, naive T cells, memory T cells, and NK cells can receive signaling via IL-2 binding to the dimeric receptor. Regulatory T cells rely on the high affinity ic receptor complex to enhance their functions, which include sequestering IL-2 away from binding to memory and naive T cells, and thereby reducing the function of these tions of cells. IL-2 mechanism of action is described in Fig. 1.

Effector T cell subsets also express the ic IL-2 receptor complex. While these cells are highly active, binding of IL-2 to a subset of effector T cells induces activation- induced cell death (AICD). Moreover, CD25 has been shown to be expressed on lung endothelium and on vascular endothelium. This expression was correlated with pulmonary edema and vascular leaking in mouse models using high dose IL-2. It was suggested that the expression of CD25 on lung cells was the reason for pulmonary toxicity of high dose IL-2 therapy. Additionally, while lung endothelial cells express CD25 under steady-state conditions, sion levels of CD25 on these cells increases in viva upon injection of mice with IL-2. It has been shown that knocking out CD25 on mune cells or interfering with the CD25 binding epitope of IL—2 by the use of immune complexes of IL-2 and anti-IL-2 antibody (IL-2/mAb) was able to t nduced pulmonary edema and vascular leak syndrome. It was also demonstrated in mice genetically modified to lack T and B cells, and sub- lethally irradiated to remove the remaining immune cells (NK, monocytes, DC, and granulocytes), addition of high dose IL-2 resulted in significant pulmonary edema, indicating a non-immune component. id="p-14" id="p-14" id="p-14"

[0014] There has been much research on the dual role of IL-2 in the ability to clear viral infections of the lung. It has been demonstrated that IL-2 is required for the expansion of CD8+ T cell for viral clearance. It has also been shown that IL-2 can mediate lung edema. For example, it has been demonstrated in a mouse in?uenza model of in?uenza viral lung infection that memory CD4+ T cells produce high levels of IL-2 and the presence of this IL-2 worsens e. Regulatory T cells are important for reducing pathological damage to lung tissue in viral infection. It has been hypothesized and trated that one mechanism to control CD8+ effector cells by Treg is by high ty consumption of IL-2 via CD25 trimeric receptor on Tregs. This in effect removes IL-2 from the expanding effector cells, and subsequently limits the availability of effector cells and potentially reduces viral nce. It is likely that Tregs also limit the effects of IL-2 on lung endothelium by sequestering away IL-2 from CD25+ endothelial cells. The outcome may be dependent on the ratio of Teff/Treg. High levels of Teff tor T cells) may lead to viral clearance but also to excessive levels of IL-2 secreted by the immune activated cells, thus leading to lung edema. In contrast, high Treg expansion may reduce pathology of lung edema but also reduce viral clearance and lead to a prolonged viral infection.

Recent data from COVID-l9 patients suggest that higher viral loads lead to poorer outcomes; therefore, reduction in viral clearance would be associated with worse outcomes. In mouse models, the role of Tregs in reducing viral clearance was demonstrated using In?uenza A virus (IAV) ion model where mice infected with IAV showed higher levels of Tregs in the lungs, spleens and lymph nodes together with higher levels of viral load in the lungs . This was observed even 6 weeks after the initiation of the infection. It was suggested that In?uenza A induces Treg ion to avoid clearance by the immune response. To evaluate whether boosting immune response would increase clearance of IAV infection in the lungs, investigators used mice previously infected with IAV and uently ed the mice with lymphocytic choriomeningitis virus (LCMV) that triggers a vigorous cytotoxic T lymphocytes se. Extensive immune response in IAV-infected lungs also led to ary edema and extensive lung tissue damage. Protection from severe pulmonary edema was achieved by treating IAV bearing mice with anti-CD25 blocking antibody prior to administration of LCMV. These data demonstrate that boosting immune response in a situation where Tregs have slowed viral clearance can induce viral clearance. In addition, it demonstrates that blocking IL-2 binding to CD25+ cells reduces the risk of immune mediated pulmonary edema during viral clearance. id="p-16" id="p-16" id="p-16"

[0016] IL-2 given as single agent therapy has been shown to enhance antiviral immune responses.

Examining the effect of IL-2 therapy during the expansion, contraction and memory phase of T cells in LCMV-infected mice demonstrated that IL-2 treatment during the expansion phase was detrimental to the survival of y dividing effector T cells that have transiently up-regulated the expression of CD25. These effector T cells were subsequently directed to AICD. In contrast, IL-2 therapy was highly cial during the contraction phase and resulted in virus-specific T cells survival and activation. It was observed that IL-2 treatment enhanced activation and proliferation of resting memory s. However, IL-2 therapy has its disadvantages. The half-life of IL-2 is short, thus multiple administrations are required, for example a daily loading dose followed by weekly dosing, leading to the risk of onal d adverse events and increased genicity. In on, the administration of exogenous high dose IL-2 would be expected to bind to CD25 positive endothelial cells. Indeed, pulmonary edema and vascular leak syndrome are the main severe adverse events for high dose IL-2 therapy in oncology. Developing technologies to overcome these limitations is critical to the use of IL-2 as a therapy.

One ofordinary skill in the art would recognize that the principles discussed above with regard to IL-2 and treating viral ions would equally apply to IL-2 and treating bacterial infections, or treating cancer.

Advances in the field of biomolecular engineering present researchers with unprecedented opportunities to apply molecular design strategies to modify naturally occurring ns and generate new molecules for targeted disease therapy. In one area, the development of immunotherapeutics such as cytokine-based or antibody-based drugs has been empowered by evolving technologies and insights from protein ering. Thus, there is a need to develop ered anti-IL-2 antibodies that would be used to modulate the functions of IL-2 in certain disease states, for example but not limited to viral or bacterial infections, and cancer.

SUMMARY id="p-19" id="p-19" id="p-19"

[0019] In one aspect, disclosed herein is an ed anti-IL-2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises heavy chain complementarity determining regions (HCDRs) HCDRl, HCDR2 and HCDR3, said VL comprises light chain complementarity determining s (LCDRs) LCDRl, LCDR2 and LCDR3, wherein said CDRs have the amino acid sequences of (a) the HCDRl comprises the amino acid sequence of SEQ ID N0238, the HCDR2 ses the amino acid sequence of SEQ ID N0239, the HCDR3 comprises the amino acid sequence of SEQ ID NO:40, the LCDRl comprises the amino acid ce of SEQ ID NO:41, the LCDR2 comprises the amino acid sequence of SEQ ID N0242, the LCDR3 comprises the amino acid sequence of SEQ ID N0243; (b) the HCDRl comprises the amino acid sequence of SEQ ID NO:44, the HCDR2 comprises the amino acid sequence of SEQ ID NO:45, the HCDR3 comprises the amino acid sequence of SEQ ID N0246, the LCDRl comprises the amino acid sequence of SEQ ID NO:47, the LCDR2 comprises the amino acid sequence of SEQ ID N0248, the LCDR3 comprises the amino acid sequence of SEQ ID N0249; (c) the HCDRl comprises the amino acid sequence of SEQ ID NO:50, the HCDR2 ses the amino acid ce of SEQ ID NO:51, the HCDR3 comprises the amino acid sequence of SEQ ID NO:52, the LCDR1 comprises the amino acid sequence of SEQ ID NO:53, the LCDR2 comprises the amino acid sequence of SEQ ID NO:54, the LCDR3 comprises the amino acid sequence of SEQ ID NO:55; (d) the HCDR1 comprises the amino acid sequence of SEQ ID NO:56, the HCDR2 comprises the amino acid sequence of SEQ ID NO:57, the HCDR3 comprises the amino acid ce of SEQ ID NO:58, the LCDR1 comprises the amino acid sequence of SEQ ID NO:59, the LCDR2 comprises the amino acid sequence of SEQ ID NO:60, the LCDR3 comprises the amino acid sequence of SEQ ID NO:61; or (e) the HCDR1 comprises the amino acid sequence of SEQ ID NO:62, the HCDR2 comprises the amino acid sequence of SEQ ID NO:63, the HCDR3 comprises the amino acid sequence of SEQ ID NO:64, the LCDR1 comprises the amino acid sequence of SEQ ID NO:65, the LCDR2 comprises the amino acid sequence of SEQ ID NO:66, the LCDR3 comprises the amino acid sequence of SEQ ID NO:67. [0019a] In another aspect, disclosed herein is an isolated anti-IL-2 dy comprising a heavy chain variable region (VH) and a light chain variable region (VL), n said VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, said VL ses light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein said CDRs have the amino acid sequences of the HCDR1 comprises the amino acid sequence of SEQ ID NO:62, the HCDR2 comprises the amino acid sequence of SEQ ID NO:63, the HCDR3 comprises the amino acid sequence of SEQ ID NO:64, the LCDR1 comprises the amino acid sequence of SEQ ID NO:65, the LCDR2 comprises the amino acid ce of SEQ ID NO:66, the LCDR3 comprises the amino acid sequence of SEQ ID NO:67.

In a related aspect, the VH and VL have the amino acid sequences of: (a) the VH ses the amino acid sequence of SEQ ID NO:10, the VL comprises the amino acid sequence of SEQ ID NO:11; (b) the VH comprises the amino acid sequence of SEQ ID NO:12, the VL comprises the amino acid sequence of SEQ ID NO:13; (c) the VH comprises the amino acid sequence of SEQ ID NO:14, the VL comprises the amino acid sequence of SEQ ID NO:15; (d) the VH ses the amino acid sequence of SEQ ID NO:16, the VL comprises the amino acid sequence of SEQ ID NO:17; 20134079_1 (GHMatters) P119595.NZ (e) the VH ses the amino acid sequence of SEQ ID NO:18, the VL comprises the amino acid sequence of SEQ ID NO:19; (f) the VH comprises the amino acid sequence of SEQ ID NO:20, the VL comprises the amino acid sequence of SEQ ID NO:21; (g) the VH comprises the amino acid sequence of SEQ ID NO:22, the VL comprises the amino acid sequence of SEQ ID NO:23; (h) the VH comprises the amino acid sequence of SEQ ID NO:24, the VL comprises the amino acid sequence of SEQ ID NO:25; (i) the VH comprises the amino acid ce of SEQ ID NO:26, the VL comprises the amino acid sequence of SEQ ID NO:27; or (j) the VH ses the amino acid sequence of SEQ ID NO:36, the VL comprises the amino acid sequence of SEQ ID NO:37.

In r related aspect, the antibody comprises an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, a F(ab')2, a minibody, a diabody, a triabody, a dy, or a single domain antibody. id="p-22" id="p-22" id="p-22"

[0022] In a r related aspect, the antibody comprises a heavy chain sing a mutation that reduces binding to Fc? or. In still a further related aspect, the antibody comprising a heavy chain sequence and a light chain sequence, (a) said heavy chain sequence set forth in SEQ ID NO: 68 and said light chain sequence set forth in SEQ ID NO: 69; (b) said heavy chain sequence set forth in SEQ ID NO: 70 and said light chain sequence set forth in SEQ ID NO: 71; or (c) said heavy chain sequence set forth in SEQ ID NO: 72 and said light chain sequence set forth in SEQ ID NO: 73.

In a related aspect, disclosed herein is a composition comprising the an anti-IL-2 antibody as described herein and a pharmaceutically acceptable carrier. In further related aspect, the composition is formulated to be at a pH between about pH 5.0 - 6.0 and comprises a buffer selected from a histidine buffer and a citrate buffer. In still a further related aspect, the composition further sing at least one of sucrose, nine, or PS80, or any combination thereof. In yet a further related aspect, the composition further comprises IL-2. id="p-24" id="p-24" id="p-24"

[0024] In one aspect, provided herein is an isolated polynucleotide sequence encoding the heavy chain variable region of an anti-IL-2 antibody, wherein the VH amino acid sequences is set forth in 20134079_1 ters) P119595.NZ the amino acid sequence of SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, or 36. In a related aspect, provided herein is a vector comprising the polynucleotide sequence ng a VH amino acid sequence set forth in the amino acid ce of SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, or 36.

In still another related aspect, provided herein is a host cell comprising the vector sing the polynucleotide sequence encoding a VH amino acid sequence set forth in the amino acid sequence of SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, or 36.

In one aspect, provided herein is an ed polynucleotide sequence encoding the light chain variable region of an anti-IL-2 antibody, wherein the VL amino acid sequences is set forth in the amino acid sequence of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, or 37. In a d aspect, provided herein is a vector comprising the cleotide sequence encoding a VL amino acid sequence set forth in the amino acid sequence of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, or 37. In still another related aspect, provided herein is a host cell comprising the vector sing the polynucleotide sequence encoding a VL amino acid sequence set forth in the amino acid sequence of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, or 37. [0025a] In r aspect, disclosed herein is an isolated polynucleotide sequence encoding the heavy chain variable region (VH) of an anti-IL-2 antibody, wherein the VH amino acid sequence is set forth in SEQ ID NO:26, and the light chain variable region (VL) of an anti-IL-2 antibody, wherein the VL amino acid sequence is set forth in SEQ ID NO: 27.

In one aspect, provided herein is an isolated cleotide sequence encoding an anti-IL-2 scFv, wherein the polynucleotide sequence is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 31, 32, 33, 34, or . In a d aspect, provided herein is a vector comprising the polynucleotide sequence encoding the scFv, wherein the polynucleotide sequence is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 31, 32, 33, 34, or 35.. In still another related aspect, provided herein is a host cell comprising the vector comprising the polynucleotide sequence encoding the scFv, wherein the polynucleotide sequence is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 31, 32, 33, 34, or 35.

[PAGE 9a TO FOLLOW] 20134079_1 (GHMatters) P119595.NZ [0026a] In another aspect, disclosed herein is an isolated polynucleotide sequence encoding an anti- IL-2 scFv, wherein the polynucleotide sequence is set forth in SEQ ID NO: 35.

In one aspect, disclosed herein is a method of treating a disease or a ion in a subject, sing the step of administering to the subject a composition comprising an anti-IL-2 antibody as described in detail herein, wherein said antibody promotes differential growth of subsets of immune cells and decreases undesirable effects caused by IL-2, thereby treating said disease or condition in said subject.

In a related aspect, the composition comprises the anti-IL-2 antibody and IL-2, or the L- 2 dy complexed with IL-2. id="p-29" id="p-29" id="p-29"

[0029] In another related aspect, the disease comprises a viral infection, a bacterial infection, or a cancer. In a further related aspect, the viral infection is caused by SARS CoV-2; norovirus; rotavirus; tis virus A, B, C, D, or E; rabies virus; West Nile virus; enterovirus; echovirus; kievirus; herpes simplex virus (HSV); HSV-2; varicella-zoster virus; to-borne viruses; arbovirus; St.

Louis encephalitis virus; California encephalitis virus; lymphocytic meningitis virus; human immunodeficiency virus (HIV); poliovirus; zika virus; rubella virus; cytomegalovirus; human papillomavirus (HPV); virus D68; severe acute respiratory syndrome (SARS) coronavirus; Middle East respiratory syndrome coronavirus; Epstein-Barr virus; nza virus; respiratory syncytial virus; polyoma viruses including JC virus; BK virus); Ebola virus; Dengue virus; or any combination thereof.

[PAGE 10 TO FOLLOW] 20134079_1 (GHMatters) P119595.NZ In another related aspect, the condition comprises a weak immune system and said treatment prophylactically boosts the immune system, or said condition comprises IL-2 induced pulmonary edema or IL-2 induced vascular leakage.

In another related aspect, the condition comprises a genetic position that increases likelihood of cancer in said t. In a further related aspect, the genetic position comprises a change in expression or activity of a gene product, said gene sing a tumor ssor gene or a mismatch repair (MMR) gene, or a combination thereof. In another further related aspect, the genetic predisposition comprises a change in expression or activity of a gene product, said gene comprising BRCAl, BRACZ, MLHl, MSH2, MSH6, PMS l, PMSZ, TP53, or CHEK2, or a combination thereof. id="p-32" id="p-32" id="p-32"

[0032] In another related aspect, the immune cells comprise one or more of naive T cells, memory T cells, CD8+ T cells, NK cells, or Natural Killer T cells. [003 3] In yet another related aspect, the undesirable effect caused by IL-2 ses one or more of activation of regulatory T cells, apoptosis of CD25+ T effector cells, IL-2 induced ary edema, pneumonia, or ILinduced vascular leakage. id="p-34" id="p-34" id="p-34"

[0034] In a further related aspect, the anti-IL-2 antibody inhibits IL-2 binding to CD25.

In yet another further related , the subject is further d with one or more immune checkpoint inhibitors targeting one or more immune checkpoints. In still a further related , the subject is treated with said immune checkpoint inhibitors concurrently, before, or after ent with said anti-IL-2 antibody. In another related aspect, the immune checkpoint comprises PD-l, PDL-l, CTLA—4, TIGIT, TIM—3, B7—H3, CD73, LAG3, CD27, CD70, 4—1BB, GITR, 0X40, SIRP—alpha (CD47), CD39, ILDR2, VISTA, BTLA, or VTCN—l.

In one aspect, provided herein is a method of immunizing a subject, wherein said immunization comprises stration of a vaccine comprising an adjuvant, said adjuvant comprising an IL-2 antibody adjuvant, said anti-IL-2 antibody comprising an anti-IL-2 dy as disclosed . In a related aspect, the IL-2 antibody adjuvant comprises the anti-IL-2 antibody and IL-2, or comprises an anti-IL-2 antibody complexed with IL-2. In a further related aspect, the subject has a weakened immune system.

In a related aspect, a method of immunization ses immunizing a subject suffering from a condition comprises a genetic predisposition that increases likelihood of cancer in said subject. In a further related , the genetic predisposition comprises a change in eXpression or activity of a gene product, said gene comprising a tumor suppressor gene or a mismatch repair (MMR) gene, or a combination thereof. In another further related aspect, the genetic predisposition comprises a change in expression or activity of a gene product, said gene comprising BRCAl, BRAC2, MLHl, MSH2, MSH6, PMSl, PMS2, TP53, or CHEK2, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS The patent or patent application file contains at least one g executed in color. Copies of this patent or patent application publication with color drawing(s) will be ed by the Office upon request and payment of the necessary fee.

The present disclosure of engineered anti-IL-2 antibodies, both as to their generation and method of use, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the anying drawings in which: Presents a tic representation of IL-2 mechanism of action and its dual role in controlling immune response.

Presents a schematic representation of anti-IL-2 antibodies-directed immunotherapy.

FIGS. 3A and 3B present a schematic representation of the progression of COVID-l9 infection and potential anti-IL-2 therapy as an adjuvant intervention. Fig. 3A is adapted from Shi Y et al., (2020) COVID-19 infection: the perspectives on immune responses. Cell Death & Differentiation volume 27, pagesl451—l454 (doi:10.1038/s4l4180530—3), Fig. l.

FIGS. 4A-4D t representative SPR sensorgram of JES6.1 antibody binding to human IL-2 (), mouse IL-2 (), and of JES6.1RMC antibody g to human IL-2 ( 4C) and mouse IL-2 ().

FIGS. SA-SC present results of IL-2 binding of YSD clones expressing JES6.1 antibody in scFv format. X axis ?uorescence levels correspond to scFv expression level of Jes6.l, Y axis ?uorescence levels correspond to binding of human or mouse IL-2. shows negative l without IL-2. shows JES6.1 YSD clones with 1000 nM human IL-2. shows YSD expressing mouse IL-2 incubated with 100nM labeled JES6.1. presents g of isolated yeast-surface display clones to IL-2 (0.1 nM). Mean Fluorescence Intensity (Em 655nM) was ized to the yeast surface expression levels. Negative YSD clones were labeled with 500nM hIL-2. presents non-specific binding ofYSD clones to mixture of OX40/PD- l/TNFR2. The clones were d with the 500nM mixture. TNFR2 binding yeast clone served as positive control. shows purification of 023 IgG. The antibody was run on a GE superdex 200 /300 increase (CV=25ml) in PBS buffer at 0.5ml/min. The leading peak (0.38CV) corresponds to a typical of aggregate, and a second peak (0.51CV) with retention of approximately 12.9ml is l of an ordinary human IgG.

FIGS. 8A-8B present binding kinetic of BDG17.023 IgG to hIL-2 () and mIL-2 (FIG. presents receptor discrimination by 023-IL-2 complex with traces of SPR se. BDG17.023 was immobilized to the CM5 chip, and hIL-2 (60 RU), CD122 (20RU) and CD25 (0RU) were streamed as indicated with an arrow.

FIGS. 10A-10D present spleen immune cell populations of mice treated with JES6.1-mIL-2 complex and BDG17.023-hIL-2 complex. Fig. 10A shows percentages of immune cell populations from mice treated with JES6.1-mIL-2 complex. Fig. 10B shows memory phenotype effector T cells (MP) CD8+/Tregs ratios of mice treated with JES6.1-mIL-2 complex. Fig. 10C shows percentages of immune cell populations from mice treated with BDG17.023-hIL-2 complex. Fig. 10D shows MP CD8+/Tregs ratios of mice treated with BDG17.023-hIL—2 x. id="p-51" id="p-51" id="p-51"

[0051] presents the alignment of amino acid sequences of the heavy chain variable region ofJES6. 1, clone 1 (17.021), clone 2 (17.022), clone 4 3), clone 5 (17.030), and clone 6 (17.035). ts the alignment of amino acid sequences of the light chain variable region of JES6. 1, clone 1 (17.021), clone 2 (17.022), clone 4 (17.023), clone 5 (17.030), and clone 6 (17.035). [005 3] FIGS. 13A and 13B present the alignment of amino acid sequences of the heavy chain variable region (A) and the light chain variable region (B) of the humanized clone 17.014, clone 17.038, clone 17.043, clone , and clone . Black triangles denote IMGT CDR ons. Bold?talic font denotes ABR/CDR positions, respectively.

FIGS. 14A-14G. Binding kinetics of indicated antibodies to human IL-2. Surface plasmon resonance (SPR) sensogram traces of binding kinetics of anti-IL-2 antibody clones BDG17.038 ( 14A), BDG (B), BDG17.053(C), 17.054(D), BDG17.066(E), BDG17.067(F), and BDG17.069(G), to human IL—2. BDG 17.038, BDG 17.043, BDG 17.066, BDG 17.067 and BDG 17.069 binding kinetics were determined by the multi- cycle method. BDG 17.053 and BDG 17.054 binding kinetics were ined by the single-cycle method. id="p-55" id="p-55" id="p-55"

[0055] FIGS. ISA-15B. Binding cs of indicated antibodies to cynomolgus monkey IL-2.

Surface plasmon resonance (SPR) sensogram traces of g kinetics of L-2 antibody clones BDG17.067 (A), and BDG17.069 (B), to cynomolgus monkey IL-2.

FIGS. 16A-16G. Differential Scanning Fluorimetry (DSF) analysis of the indicated IgGs’ melting point. light green dashed line indicates Tonset and bold green dashed lines indicate Tm1, and where applicable Tm2. Anti-IL-2 clones analyzed are BDG17.038 (A), BDG17.043(B), BDG 17.053 (C), BDG 17.054 (D), 066 (E), BDG17.067 (F), and BDG17.069 (G).

FIGS. 17A-17B. Present receptor discrimination of ted antibody/IL-2 complex by g of SPR response. Antibody was immobilized to the CM5 chip, and hIL-2, CD 122, and CD25 were streamed as indicated with an arrow. A presents a schematic order of injection of compounds to SPR chip, that represents sequential anti-IL-2 antibodies complexed with human IL- 2 (hlL2), binding to CD122 but not to CD25. FIGS. 17B-17G present the SPR se: BDG17.038 (B), BDG017.043 (C), 17.054 (D), BDG17.066 (E), BDG17.067 (F), and BDG17.069 (G).

FIGS. 18A and 18B show anti-human IL-2 dies (clones 17.043 and 17.054) demonstrate potent immune stimulating effect in vivo. The anti-IL-2 antibody/hIL-2 xes increase effector cell populations with no observed effect on regulatory T cells. A shows C57BL/6 mice were administered daily with L-2 antibody (10ug) pre-complexed with 0.5ug of hlL-2 for four days. B shows C57BL/6 mice were administered daily with anti-IL-2 antibody (25ug) pre-complexed with 1.25ug of hlL-2 for four days. On day 5, splenocytes were isolated and immune cell populations were analyzed by ?ow cytometry. Presented are mean values for each experimental group (n=6 per group). Lymphocytes were gated according to side-scatter and forward- scatter parameters and subsequent immune cells subpopulations were gated as follows: Tregs (CD45+, CD3+, CD4+, CD25+, FoxP3+), CD8 T cells (CD45+, CD3+, CD8+, CD122+, CD25—), NKT—cells (CD45+, CD3+, CD49b+, NK1.1+), NK cells (CD45+, CD3—, , NK1.1+). id="p-59" id="p-59" id="p-59"

[0059] FIGS. 19A and 19B show anti-human IL-2 antibodies (clones 17.043 and 17.054) demonstrate potent dose ent immune stimulating effect in vivo. A shows C57BL/6 y mice were administered daily with anti-IL-2 dy/hIL-2 complex 1.25ug respectively) for four days. On day 5, splenocytes were isolated and immune cells populations were ed by ?ow try. B shows anti-human IL-2 antibodies demonstrate potent in vivo immune stimulating effect in a dose dependent manner. C57BL/6 healthy mice were administered daily with increasing doses of anti-IL-2 antibody/hIL-2 complex as indicated. On day 5, splenocytes were isolated and immune cells populations were analyzed using flow cytometry. Lymphocytes were gated according to side-scatter and forward-scatter parameters and subsequent immune cells ulations were gated as follows: Tregs (CD45+, CD3+, CD4+, CD25+, FoxP3+), CD8 T cells (CD45+, CD3+, CD8+, , CD25—), NKT—cells , CD3+, CD49b+, NK1.1+), NK cells , CD3—, CD49b+, NK1.1+).

FIGS. 20A and 20B show anti-human IL-2 dies (clones 17.043 and 17.054) demonstrate safe dose regimen in vivo. A shows C57BL/6 healthy mice were administered daily with anti-IL-2 antibody/hIL-2 complex (10ug/0.5ug respectively) for four days. B shows C57BL/6 healthy mice were administered daily with anti-IL-2 antibody/hIL-2 complex (25ug/1.25ug respectively) for four days. At the end of the experiments, mice were weighed and percent of body weight changes were calculated in respective to the weight of each mouse at the beginning of the study. Presented are mean percent of body weight (BW) change for each experimental group (n=6 per group).

FIGS. 21A and 21B show anti IL-2 antibodies (clones 17.043 and 17.054) inhibit tumor growth in an I/O resistant tumor model with a tolerable safety profile. C57BL/6 y mice were inoculated with B16F10 melanoma tumor cells on day 0. On day 5, the mice were randomized to experimental groups (n=10 per group) and administered daily with anti-IL-2 antibody/hIL-2 complex (20ug/1ug respectively) or with PBS for four days. A shows changes in tumor volume for each experimental group. B shows changes in body weight for each experimental group. t body weight changes were calculated in respective to the weight of each mouse at the beginning of the study.

FIGS. 22A-22G show the results of ing the different formulations of anti-IL-2 antibody clone BDG 17.069. A presents the BDG 17.069 parameters at T=0. B presents BDG 17.069 appearance, pH, protein concentration, and sub-visual particle formation at T=0 and after incubation at 40°C for 1 and 2 weeks. C presents BDG 17.069 SEC, caliper-SDS and ary isoelectric ng analysis at T=0 and after incubation at 40°C for 1- and 2-weeks. D presents BDG 17.069 appearance, pH, protein concentration, and sub-visual particle formation at T=0 and post agitation at 300rpm for 3 days. E presents BDG 17.069 SEC, caliper- SDS, and capillary isoelectric focusing analysis at T=0 and post agitation at 300rpm for 3 days. F presents BDG 17.069 appearance, pH, n concentration, and sub le formation at T=0 and after five cycles of /Thaw. G presents BDG 17.069 SEC, caliper- SDS, and capillary isoelectric focusing analysis at T=0 and after five cycles of Freeze/Thaw.

DETAILED DESCRIPTION The present disclosure provides engineered anti-human IL-2 antibodies that bind human IL- 2 with high ty (e.g., l2.7pM to 48pM) to a pre-def1ned binding epitope. The antibodies bind to IL-2 in a manner that completely prevents CD25 binding, yet spares the binding of IL-2 to CD122, thereby modulating immune ses towards immune stimulation by directly activating and expanding effector cells without cting with CD25-expressing cells (e.g., regulatory T-cells, short lived cytotoxic T-cells, pulmonary endothelial cells and vascular endothelial . Thus, the antibody/IL-2 complex would drive a robust immune response to clear viral load or tumor by ing and activating effector cells such as NK cells, central memory T cells and virus or tumor- specific T-cells while inhibiting IL-2 activation induced cell death of the short lived CD25+cytotoxic T-cells that are important for viral/tumor nce. The antibody?L-2 complex would also decrease immunosuppression caused by the regulatory arm of the immune system. Moreover, the antibodyHL- 2 complex would prevent undesired interactions of IL—2 with vascular and pulmonary CD25- expressing cells, thereby preventing severe syndromes of IL-2 induced vascular leakage and IL-2 induced pulmonary edema frequently seen in models of viral lung infections. In some embodiments, the activity of the engineered anti-IL-2 antibodies described herein is dependent on the pre-defined epitope to which they are designed to bind.

A skilled artisan would appreciate that in certain embodiments, the term "anti-IL-2 dy" as used herein is interchangeable with the term "anti-human-IL-2 antibody", having all the same qualities and gs. Similarly, as used throughout, in certain embodiments, the term "IL-2" is interchangeable with the term "human IL-2", having all the same qualities and gs.

In some embodiments, an anti-human IL-2 antibody described herein inhibits binding of IL- 2 with an IL-2 or alpha (IL-2 Ra, i.e., CD25) subunit and therefore ts binding to the trimer IL-2 RaBry receptor. In certain embodiments, anti-IL-2 dies that inhibit binding of IL-2 with a trimer IL-2 receptor (IL-2 RaBy) do not inhibit binding of IL-2 with the dimer IL-2 receptor (IL-2 presents a schematic of anti-IL-2 dies directed therapy. Targeting IL-2 to different cell populations can be used to either modulate the immune response toward immunosuppression or towards immune activation. The anti-human IL-2 antibodies disclosed herein are designed to bind with high affinity to an IL-2 epitope that blocks IL-2 binding to CD25. As a , IL-2 is ted from binding to short-lived CD8+ cytotoxic T cells or regulatory T cells that express high level of CD25 but is redirected to preferentially bind to effector T cells to stimulate enhanced immune response to improve viral or bacterial clearance. Moreover, since IL-2 binding to CD25-expressing endothelial cells is also blocked, IL-2 induced ary edema and vascular leaking would also be prevented.

In one embodiment, the present disclosure provides a method of treating a e (e.g., viral infection, bacterial ion, or ), or a condition (e. g., an undesirable condition caused by IL- 2, for example but not limited to lung edema) with an anti-IL-2 antibody designed to enhance T cell immune response and to prevent severe edema ms of acute pneumonia induced by IL-2. The anti-IL-2 antibody would bind specifically to human IL-2 with high affinity at a pre-def1ned epitope that blocks IL-2 binding to the alpha chain of the IL-2 receptors (CD25) while sparing binding to the main signaling beta chain and gamma chain complex of the receptor (CDl22/CD132). Consequently, in the presence of such antibody, IL-2 would be directed to immune cells responsible for viral/tumor nce and away from cells that slow the immune response or cause the edema. The formation of this IL-2/antibody immunocomplex will direct IL-2 to bind and activate exclusively naive and memory T lymphocytes, NK cells, and Natural Killer T lymphocytes while ting activation of regulatory T cells and apoptosis of short-lived CD25+ cytotoxic T effector cells. Altogether, the end result is an effective immune response, for example, viral or tumor clearance. In addition, this treatment will prevent the ty caused by IL—2 binding to endothelial CD25 expressing cells. Thus, in one embodiment, ing IL-2 with the anti-IL-2 antibodies disclosed herein would be an effective treatment for respiratory diseases caused by viral or bacterial infections. In r embodiment, ent with the anti-IL-2 antibodies disclosed herein would be effective in preventing the toxicity caused by IL-2 binding to endothelial CD25 expressing cells, e.g., pulmonary edema, or nduced vascular leakage. More importantly, the enhancement of IL-2 immune stimulation towards general immune activation and expansion of immune effector cells independent of a specific pathogen (e.g., a viral antigen) would be an effective strategy against future viral or bacterial pandemics caused by an unknown pathogen (Figs. 3A and 3B).

In one embodiment, the method disclosed herein would be useful t infection caused by SARS Co-V2. The SARS Co-V2 binds to angiotensin converting enzyme 2 of lung cells that allow for viral entry and replication. The immune response to viral infections of the lung consists of both the innate and acquired arms of the immune system. As in the cases with many respiratory viruses, clearance of oV2 from the lung is expected to be dependent on T cell immune response. The cytokine IL-2 is critical for the expansion of T cells and plays an important role in immune responses to viruses. However, in addition to its pro-stimulatory role IL-2 also induces some adverse side effects like lung edema and vascular leak syndrome h its binding to endothelium expressing the CD25 receptor.

FIGS. 3A and3B presents a schematic of the progression of COVID-l9 infection and potential anti-IL-2 therapy as an adjuvant intervention. Fig. 3A shows the invading SARS Co-V2 causes non-severe ms and s protective immune responses after an incubation period.

Successful elimination of the infection relies on the health status of the infected individual. Individuals with poor immune responses to the virus would have difficulty in clearing the virus while individuals with an over robust immune response may lead to pulmonary edema and other cytokine mediated e effects. Therefore, strategies that boost immune response and prevent pulmonary edema are desired. While high concentrations of IL-2 would be beneficial for viral clearance, particularly at the early stage, high levels of IL-2 could lead to ILinduced pulmonary edema and vascular leaking through interactions between IL-2 and CD25-expressing endothelial cells. Fig. 3B shows that an anti- human IL-2 dy designed to bind and block the CD25HL-2 ction is predicted to enhance ion of immune effector cells to improve viral clearance and reduce the negative effects of IL- 2 g to CD25 expressed on endothelial cells, thereby preventing IL-2 induced ary edema and ar leaking. id="p-70" id="p-70" id="p-70"

[0070] presents a schematic for the ism of action of IL-2 and its dual role in controlling immune response. The left panel shows IL-2 consists of three binding epitope sites (on, B, y) that interact with different forms of IL2-R (CD25, CD122 and CD132) with different affinities. The right panel shows different IL-2R complexes are expressed on different T cell populations, and their different affinities to IL2 allow immunosuppression under conditions of low local concentrations of IL-2 and immune stimulation when IL-2 local concentration rises.

In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the antibodies disclosed herein. However, it will be understood by those d in the art that preparation and uses of antibodies disclosed herein may in n cases be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to e the disclosure presented herein.

Throughout this application, various references or publications are cited. Disclosures of these references or publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

As used herein, the term "antibody" may be used interchangeably with the term "immunoglobulin", having all the same qualities and meanings. An antibody binding domain or an antigen binding site can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in specifically g with a target antigen. By "specifically binding" is meant that the binding is selective for the antigen of interest and can be discriminated from unwanted or cific interactions. For e, an antibody is said to ically bind an IL-2 epitope when the equilibrium dissociation constant is 5 105, 106, or 10'7 M.

In some embodiments, the equilibrium dissociation constant may be 5 10'8 M or 10'9 M. In some r embodiments, the equilibrium dissociation constant may be 5 10'10 M, 10'11 M, or . In some embodiments, the brium dissociation constant may be in the range of 5 10'5 M to 10'12M.

As used , the term "antibody" encompasses an antibody fragment or fragments that retain binding specificity including, but not d to, IgG, heavy chain variable region (VH), light chain variable region (VL), Fab fragments, F(ab‘)2 fragments, scFv fragments, Fv fragments, a nanobody, minibodies, diabodies, triabodies, tetrabodies, and single domain antibodies (see, e.g., Hudson and Souriau, Nature Med. 9: 129-134 (2003)). Also encompassed are humanized, ized, and chimeric antibodies as these terms are generally understood in the art.

As used , the term "heavy chain variable region" may be used hangeably with the term "VH domain" or the term "VH", having all the same meanings and qualities. As used herein, the term "light chain variable region" may be used interchangeably with the term "VL domain" or the term "VL", having all the same meanings and qualities. A skilled artisan would recognize that a "heavy chain variable region" or "VH" with regard to an antibody encompasses the fragment of the heavy chain that contains three complementarity determining s (CDRs) interposed between g stretches known as framework regions. The framework regions are more highly conserved than the CDRs, and form a scaffold to support the CDRs. Similarly, a d artisan would also recognize that a "light chain variable region" or "VL" with regard to an antibody encompasses the fragment of the light chain that contains three CDRs osed between framework regions.

As used herein, the term "complementarity determining region" or "CDR" refers to the hypervariable region(s) of a heavy or light chain variable region. Proceeding from the N—terminus, each of a heavy or light chain polypeptide has three CDRs denoted as "CDRl," "CDR2," and "CDR3". Crystallographic analysis of a number of n-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with a bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the CDR s are primarily responsible for the specificity of an antigen-binding site. In one embodiment, an antigen-binding site includes six CDRs, comprising the CDRs from each of a heavy and a light chain variable region.

As used , the term "framework region" or "FR" refers to the four ?anking amino acid sequences which frame the CDRs of a heavy or light chain variable region. Some FR residues may contact bound antigen; however, FR residues are primarily responsible for folding the variable region into the n-binding site. In some ments, the FR residues sible for folding the variable regions comprise residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural es are very highly conserved. In this regard, all variable region sequences contain an internal disulfide loop of around 90 amino acid es. When a variable region folds into an antigen binding site, the CDRs are displayed as projecting loop motifs that form an antigen- binding surface. It is generally recognized that there are conserved structural regions of FR that in?uence the folded shape of the CDR loops into certain "canonical" ures regardless of the precise CDR amino acid sequence. Furthermore, certain FR residues are known to participate in non- covalent interdomain contacts which stabilize the ction of the antibody heavy and light chains.

Wu and Kabat (Tai Te Wu, Elvin A. Kabat. An analysis of the sequences of the variable regions of bence jones proteins and myeloma light chains and their ations for antibody mentarity. Journal of Experimental Medicine, 132, 2, 8 (1970); Kabat EA, Wu TT, Bilofsky H, Reid-Miller M, Perry H. Sequence of proteins of immunological interest. Bethesda: National Institute of Health; 1983. 323 ) pioneered the alignment of antibody peptide sequences, and their contributions in this regard were several-fold: Firstly, h study of sequence similarities between variable domains, they identified correspondent residues that to a greater or lesser extent were gous across all antibodies in all vertebrate species, inasmuch as they adopted similar three-dimensional structure, played similar functional roles, interacted similarly with neighboring residues, and existed in similar chemical environments. ly, they d a peptide sequence numbering system in which homologous immunoglobulin residues were assigned the same position number. One skilled in the art can unambiguously assign to any variable domain sequence what is now commonly called Kabat numbering without reliance on any experimental data beyond the ce itself. Thirdly, Kabat and Wu calculated variability for each Kabat-numbered sequence position, by which is meant the finding of few or many possible amino acids when variable domain sequences are d. They identified three contiguous regions of high variability embedded within four less variable contiguous regions. Kabat and Wu formally demarcated residues constituting these variable tracts, and designated these "complementarity determining regions" (CDRs), referring to chemical complementarity between antibody and antigen. A role in three-dimensional folding of the variable domain, but not in n recognition, was ed to the ing ariable regions, which are now termed "framework regions". Fourth, Kabat and Wu established a public database of antibody peptide and nucleic acid sequences, which continues to be maintained and is well known to those skilled in the art. id="p-79" id="p-79" id="p-79"

[0079] a and coworkers (Cyrus Chothia, Arthur M. Lesk. Canonical structures for the hypervariable regions of immunoglobulins. l of Molecular Biology, 196, 4, 8 (1987)) found that certain sub portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub portions were designated as L1, L2 and L3 or H1, H2 and H3, where the "L" and the "H" designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs.

More recent studies have shown that virtually all antibody binding residues fall within regions of structural consensus. (Kunik, V. et al., PloS Computational y 1002388 ary 2012)). In some embodiments, these s are referred to as antibody binding regions. It was shown that these s can be identified from the antibody sequence as well. "Paratome", an implementation of a structural approach for the identification of structural consensus in antibodies, was used for this purpose. (Ofran, Y. et al., J. Immunol. 757:6230-6235 (2008)). While residues identified by Paratome cover virtually all the antibody g sites, the CDRs (as identified by the commonly used CDR identification tools) miss significant portions of them. dy binding residues which were identified by Paratome but were not identified by any of the common CDR identification methods are referred to as Paratome-unique residues. Similarly, antibody binding residues that are identified by any of the common CDR identification methods but are not identified by Paratome are referred to as CDR-unique residues. Paratome-unique residues make l energetic contribution to antibody-antigen interactions, while CDRs-unique residues have a rather minor contribution. These results allow for better identification of antigen binding sites.

IMGT® is the international ImMunoGeneTics information system®, (See, Nucleic Acids Res. 2015 Jan; 43 (Database issue):D4l3-22. doi: 10.1093/nar/gku1056. Epub 2014 NOV 5 Free article. PMID: 25378316 LIGM2441 and Dev Comp Immunol. 2003 Jan;27(l):55-77). IMGT is a unique numbering system for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains (Lefranc et al., Dev Comp Immunol. 27: 55-77 (2003)). IMGT® presents a uniform numbering system for these IG and TcR variable domain sequences, based on aligning 5 or more IG and TcR variable region sequences, taking into account and combining the Kabat de?nition ofFRs and CDRs, ural data, and Chothia‘s characterization of the hypervariable loops.

IMGT is considered well known in the art as a universal numbering scheme for antibodies.

In some embodiments, fication of potential t amino acid positions in the VH and VL domains uses the IMGT system of analysis. In some embodiments, identi?cation of potential variant amino acid positions in the VH and VL domains uses the Paratome system of analysis. In some embodiments, identi?cation of potential variant amino acid ons in the VH and VL domains uses the Kabat system of analysis. In some embodiments, fication of potential variant amino acid positions in the VH and VL domains uses the Clothia system of analysis. id="p-83" id="p-83" id="p-83"

[0083] In describing variant amino acid positions present in the VH and VL domains, in some embodiments the IMGT numbering is used. In describing variant amino acid ons present in the VH and VL domains, in some embodiments the Paratome numbering is used. In describing variant amino acid positions t in the VH and VL domains, in some embodiments the Kabat numbering is used. In describing variant amino acid positions present in the VH and VL domains, in some embodiments the Clothia numbering is used.

Antigen binding sequences are conventionally located within the heavy chain and light chain variable s of an antibody. These heavy and light chain variable regions may, in certain instances, be manipulated to create new binding sites, for example to create antibodies or fragments thereof, that bind to a ent antigen or to a different epitope of the same n. In some embodiments, as bed herein, manipulating the sequences of a heavy chain variable region or the sequences of a light chain le , or both, would create a new g site for a second An antibody may eXist in various forms or having various domains including, without limitation, a complementarity determining region (CDR), a variable region (Fv), a VH domain, a VL domain, a single chain variable region (scFv), and a Fab fragment.

A person of ordinary skill in the art would appreciate that a scFv is a fusion polypeptide comprising the variable heavy chain (VH) and variable light chain (VL) regions of an immunoglobulin, connected by a short linker e, the linker may have, for example, 10 to about amino acids.

A skilled artisan would also appreciate that the term "Fab" with regard to an antibody generally encompasses that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond, whereas F(ab’)2 comprises a fragment of a heavy chain comprising a VH domain and a light chain comprising a VL .

In some embodiments, an antibody encompasses whole dy molecules, including monoclonal and polyclonal antibodies. In some embodiments, an antibody encompasses an antibody fragment or nts that retain g specificity including, but not limited to, variable heavy chain (VH) fragments, variable light chain (VL) fragments, Fab nts, F(ab‘)2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

Engineered Anti-IL-Z Antibodies id="p-89" id="p-89" id="p-89"

[0089] In one embodiment, the present disclosure provides engineered anti-IL-2 antibodies resulted from introducing amino acid variations to a parent anti-IL-2 antibody. In one embodiment, the one or more of the amino acid variations are introduced in a CDR region. In another embodiment, the one or more amino acid variations are introduced within a framework (FR) region. In yet r embodiment, the amino acid variations are uced to both the CDR region and the framework (FR) region. One of ry skill in the art would readily employ various standard techniques known in the art to introduce amino acid variations into an anti-IL-2 antibody and then test the ing modified antibodies for any s of binding to IL-2. While standard techniques may be used, the resultant binding pattern of the new created antibodies is not predictable and must be analyzed to determine functionality. id="p-90" id="p-90" id="p-90"

[0090] In n embodiments, the present sure provides polypeptides comprising the VH and VL domains which could be dimerized under le conditions. For example, the VH and VL domains may be combined in a suitable buffer and zed through appropriate interactions such as hydrophobic interactions. In another embodiment, the VH and VL domains may be combined in a suitable buffer containing an enzyme and/or a or which can promote dimerization of the VH and VL domains. In another embodiment, the VH and VL domains may be combined in a suitable vehicle that allows them to react with each other in the presence of a suitable reagent and/or catalyst.

In certain embodiments, the VH and VL domains may be contained within longer polypeptide sequences, that may include for example but not limited to, constant regions, hinge regions, linker regions, Fc regions, or disulf1de binding regions, or any combination thereof. A constant domain is an immunoglobulin fold unit of the constant part of an globulin molecule, also referred to as a domain of the constant region (e.g., CH1, CH2, CH3, CH4, Ck, Cl). In some ments, the longer polypeptides may comprise multiple copies of one or both of the VH and VL domains ted according to the method disclosed herein; for example, when the polypeptides generated herein are used to forms a diabody or a triabody.

In some ments, the Fc region comprises at least one mutation that reduces Fc-gamma binding, i.e., binding to a ch or (chRs). In some embodiments, reduced binding is abolished binding, which binding to the ch receptor is not detectable. In some embodiments, reduced binding reduces the binding y to a ch receptor. In some embodiments, reduced binding reduces the on rate for binding to a ch receptor. In some embodiments, reduced binding reduces the off rate of binding to a ch receptor. In some ments, a mutation that reduces the Fc-gamma binding comprises L234A, L235A mutations, also known as LALA mutations. In some embodiments, a mutation that reduces the Fc-gamma binding comprises a P329G mutation in addition to the L234A, L235A mutations. In some ments, an antibody bed herein comprises a heavy chain comprising a mutation that reduces binding to ch receptor.

In one embodiment, the present disclosure provides an engineered (or modi?ed) anti-IL-2 antibody, n the antibody comprises a heavy chain variable region having the sequence of one of SEQ ID NOs: 10, l2, l4, 16, 18, 20, 22, 24, 26, or 36. In one embodiment, the engineered antibody can be an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, or a F(ab’)2. The IgG can be of the subclass of IgG1, IgG2, IgG3, or IgG4. In another embodiment, the engineered antibody can be part of a minibody, a y, a dy, a nanobody, or a single domain dy. id="p-94" id="p-94" id="p-94"

[0094] In one embodiment, the present disclosure provides an engineered (or modi?ed) anti-IL-2 antibody, n the antibody comprises a light chain variable region having the sequence of one of SEQ ID NOs211, 13, 15, l7, 19, 21, 23, 25, 27, or 37. In one embodiment, the engineered antibody can be an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, or a F(ab’)2. The IgG can be of the subclass of IgG1, IgG2, IgG3, or IgG4. In another embodiment, the engineered antibody can be part of a minibody, a diabody, a triabody, a nanobody, or a single domain antibody.

In one embodiment, the present disclosure provides an engineered (or modi?ed) anti-IL-2 antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region having the sequences of one of: SEQ ID NOs210 and 11; SEQ ID NOs212 and 13; SEQ ID NOs214 and 15; SEQ ID NOs216 and 17; SEQ ID NOs218 and 19; SEQ ID NOs22O and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27, or SEQ ID NOs: 36 and 37. In one embodiment, the engineered anti-IL-2 antibody comprises the sequences of SEQ ID NOs: 10 and 11.

In one embodiment, the engineered L-2 antibody comprises the sequences of SEQ ID NOs212 and 13. In one embodiment, the engineered anti-IL-2 antibody comprises the sequences of SEQ ID NOs214 and 15. In one ment, the ered anti-IL-2 antibody comprises the ces of SEQ ID NOs216 and 17. In one embodiment, the engineered L-2 antibody comprises the sequences of SEQ ID NOs: 18 and 19. In one embodiment, the engineered anti-IL-2 dy comprises the sequences of SEQ ID NOs22O and 21. In one embodiment, the engineered anti-IL-2 antibody comprises the sequences of SEQ ID NOs222 and 23. In one embodiment, the engineered anti-IL-2 antibody comprises the sequences of SEQ ID NOs224 and 25. In one embodiment, the engineered anti-IL-2 antibody comprises the sequences of SEQ ID NOs226 and 27. In one embodiment, the engineered anti-IL-2 antibody comprises the ces of SEQ ID NOs236 and 37.

In some embodiments, an isolated anti-IL-2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises heavy chain complementarity determining regions (HCDRs) HCDRl, HCDR2 and HCDR3, said VL comprises light chain complementarity determining regions (LCDRs) LCDRl, LCDR2 and LCDR3, wherein said CDRs have the amino acid sequences of (a) the HCDRl comprises the amino acid ce of SEQ ID NO:38, the HCDR2 comprises the amino acid sequence of SEQ ID NO:39, the HCDR3 comprises the amino acid ce of SEQ ID NO:40, the LCDRl comprises the amino acid sequence of SEQ ID NO:41, the LCDR2 comprises the amino acid sequence of SEQ ID NO:42, the LCDR3 comprises the amino acid sequence of SEQ ID NO:43; (b) the HCDRl comprises the amino acid sequence of SEQ ID NO:44, the HCDR2 comprises the amino acid sequence of SEQ ID NO:45, the HCDR3 comprises the amino acid sequence of SEQ ID N0246, the LCDRl ses the amino acid sequence of SEQ ID NO:47, the LCDR2 ses the amino acid sequence of SEQ ID N0248, the LCDR3 comprises the amino acid sequence of SEQ ID N0249; (C) the HCDRl comprises the amino acid sequence of SEQ ID NO:50, the HCDR2 comprises the amino acid sequence of SEQ ID NO:51, the HCDR3 comprises the amino acid sequence of SEQ ID N0252, the LCDRl comprises the amino acid sequence of SEQ ID N0253, the LCDR2 comprises the amino acid sequence of SEQ ID NO:54, the LCDR3 comprises the amino acid sequence of SEQ ID NO:55; (d) the HCDRl comprises the amino acid sequence of SEQ ID NO:56, the HCDR2 comprises the amino acid sequence of SEQ ID NO:57, the HCDR3 comprises the amino acid sequence of SEQ ID N0258, the LCDRl comprises the amino acid sequence of SEQ ID N0259, the LCDR2 comprises the amino acid sequence of SEQ ID NO:60, the LCDR3 comprises the amino acid sequence of SEQ ID NO:61; or (e) the HCDRl ses the amino acid ce of SEQ ID N0262, the HCDR2 comprises the amino acid sequence of SEQ ID N0263, the HCDR3 comprises the amino acid sequence of SEQ ID N0264, the LCDRl comprises the amino acid sequence of SEQ ID NO:65, the LCDR2 comprises the amino acid sequence of SEQ ID N0266, the LCDR3 comprises the amino acid sequence of SEQ ID NO:67.

In some ments, the VH and VL have the amino acid sequences wherein the VH comprises the amino acid ce of SEQ ID NO: 10, the VL comprises the amino acid sequence of SEQ ID NO:11; the VH comprises the amino acid sequence of SEQ ID NO: 12, the VL comprises the amino acid sequence of SEQ ID NO:13; the VH comprises the amino acid sequence of SEQ ID NO: 14, the VL comprises the amino acid ce of SEQ ID NO: 15; the VH comprises the amino acid sequence of SEQ ID NO: 16, the VL comprises the amino acid sequence of SEQ ID NO: 17; the VH comprises the amino acid sequence of SEQ ID NO: 18, the VL comprises the amino acid sequence of SEQ ID NO: 19; the VH comprises the amino acid sequence of SEQ ID N0220, the VL comprises the amino acid sequence of SEQ ID NO:21; the VH comprises the amino acid sequence of SEQ ID N0222, the VL comprises the amino acid sequence of SEQ ID N0223; the VH comprises the amino acid sequence of SEQ ID N0224, the VL comprises the amino acid sequence of SEQ ID N0225; the VH ses the amino acid sequence of SEQ ID N0226, the VL comprises the amino acid sequence of SEQ ID NO:27; or the VH comprises the amino acid sequence of SEQ ID N0236, the VL ses the amino acid sequence of SEQ ID N0237. id="p-98" id="p-98" id="p-98"

[0098] In some embodiments, an antibody comprising a heavy chain sequence and a light chain sequence, said heavy chain sequence set forth in SEQ ID NO: 68 and said light chain sequence set forth in SEQ ID NO: 69; said heavy chain ce set forth in SEQ ID NO: 70 and said light chain sequence set forth in SEQ ID NO: 71; or said heavy chain sequence set forth in SEQ ID NO: 72 and said light chain sequence set forth in SEQ ID NO: 73.

In one embodiment, the engineered antibody can be an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, or a F(ab’)2. The IgG can be of the subclass of IgG1, IgG2, IgG3, or IgG4. In another embodiment, the engineered antibody can be part of a minibody, a diabody, a triabody, a nanobody, or a single domain antibody.

In one embodiment, the present disclosure also provides isolated cleotide ce encoding a heavy chain variable region of an anti-IL-2 antibody, wherein the heavy chain variable region comprises the amino acid sequence of one of SEQ ID NOs210, l2, l4, 16, 18, 20, 22, 24, 26, or 36. In another ment, the present disclosure also provides a vector comprising the above- mentioned polynucleotide sequences. In view of the amino acid sequences disclosed herein, one of ordinary skill in the art would readily construct a vector or plasmid to encode for the amino acid sequences. In another embodiment, the present disclosure also provides a host cell comprising the vector ed . Depending on the uses and experimental conditions, one of skill in the art would readily employ a suitable host cell to carry and/or express the above-mentioned polynucleotide In one embodiment, the present disclosure also es isolated polynucleotide sequence ng a light chain variable region of an anti-IL-2 antibody, wherein the light chain variable region comprises the amino acid sequence of one of SEQ ID NOs:ll, 13, 15, l7, 19, 21, 23, 25, 27, or 37.

In another embodiment, the present disclosure also provides a vector comprising the above-mentioned polynucleotide sequences. In view of the amino acid sequences disclosed herein, one of ordinary skill in the art would readily construct a vector or plasmid to encode for the amino acid ces. In another embodiment, the present disclosure also provides a host cell sing the vector provided herein. Depending on the uses and experimental conditions, one of skill in the art would readily employ a suitable host cell to carry and/or express the above-mentioned cleotide sequences.

In view of the sequences for the heavy chain variable regions and light chain le regions disclosed herein, one of ordinary skill in the art would readily employ standard techniques known in the art to construct an anti-IL-2 scFv. In one embodiment, polynucleotide ces encoding for such anti-IL-2 scFv could have the sequence of one of SEQ ID NOs21-5 or one of SEQ ID NO: 31- In certain embodiments, an isolated polynucleotide sequence disclosed herein, encoding the heavy chain variable region of an L-2 antibody, comprises a VH amino acid ce set forth in the amino acid sequence of any of SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, or 36. In some embodiments, a vector comprises the polynucleotide sequence of any of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, or 36. In some embodiments, a host cell comprising the vector comprising the polynucleotide sequence of any of SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, or 36.

In certain embodiments, an isolated polynucleotide sequence disclosed herein, encoding the light chain variable region of an anti-IL-2 antibody, comprises a VL amino acid sequence as set forth in the amino acid sequence of any of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, or 37. In some embodiments, a vector ses the cleotide sequence comprising the amino acid sequence of any of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, or 37. In some embodiments, a host cell comprises a vector comprising the polynucleotide sequence encoding the amino acid sequence of any of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, or 37. In some embodiments, an isolated polynucleotide sequence encodes an anti-IL-2 scFv, wherein the polynucleotide ce is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 31, 32, 33, 34, or 35. In some embodiments, a vector comprises an isolated polynucleotide sequence encodes an anti-IL-2 scFv, wherein the polynucleotide sequence is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 31, 32, 33, 34, or 35. In some embodiments, a host cell comprises a vector comprising an isolated polynucleotide sequence encoding an anti-IL-2 scFv, wherein the polynucleotide sequence is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 31, 32, 33, 34, or 35. id="p-105" id="p-105" id="p-105"

[0105] In another embodiment, the present disclosure also provides an isolated anti-IL-2 antibody, wherein the antibody ses a heavy chain variable region having complementarity determining region (CDR) 1, CDR2 and CDR3. In one ment, the CDR1, CDR2 and CDR3 comprise amino acid ces of SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 tively; SEQ ID NOs250—52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID NOs262-64 respectively. In one embodiment, the antibody can be an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, a F(ab’)2, a minibody, a diabody, a triabody, a nanobody, or a single domain antibody. The IgG can be IgGl, IgG2, IgG3, or an IgG4. In one embodiment, the t disclosure also encompasses a composition comprising the above-mentioned dy and a pharmaceutically acceptable carrier.

In r embodiment, the t disclosure also provides an isolated anti-IL-2 antibody, wherein the antibody comprises a light chain le region having complementarity determining region (CDR) 1, CDR2 and CDR3. In one embodiment, the CDR1, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs24l-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253—55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID NOs265-67 respectively. In one embodiment, the antibody can be an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, a F(ab’)2, a dy, a diabody, a triabody, a nanobody, or a single domain antibody. The IgG can be IgGl, IgG2, IgG3, or an IgG4. In one embodiment, the present disclosure also encompasses a composition comprising the above-mentioned antibody and a pharmaceutically acceptable carrier.

In another embodiment, the present disclosure also provides an isolated L-2 antibody, wherein the antibody comprises a heavy chain variable region having complementarity determining region (CDR) l, CDR2 and CDR3, and a light chain variable region having CDRl, CDR2 and CDR3.

In one embodiment, the heavy chain CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs238-40 tively; SEQ ID NOs244-46 respectively; SEQ ID NOs250-52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID -64 respectively. In one embodiment, the light chain CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs24l-43 tively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID NOs265-67 respectively. In one ment, the antibody can be an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, a F(ab’)2, a dy, a diabody, a triabody, a nanobody, or a single domain antibody. The IgG can be IgGl, IgG2, IgG3, or an IgG4. In one embodiment, the present disclosure also encompasses a composition comprising the above-mentioned antibody and a pharmaceutically acceptable carrier.

Pharmaceutical Compositions In some ments, disclosed herein are compositions for therapeutic use. In some embodiments, a composition described herein ses an anti-IL-2 dy as sed herein and a pharmaceutically acceptable carrier.

As used herein, the terms "composition" and pharmaceutical composition" may in some embodiments, be used interchangeably having all the same ies and meanings. In some embodiments, disclosed herein is a pharmaceutical composition for the treatment of a condition or disease as bed herein.

In some embodiments, disclosed herein are pharmaceutical compositions for use in a combination therapy. id="p-111" id="p-111" id="p-111"

[0111] In another embodiment, disclosed herein are compositions for use treating a disease or condition in a subject. In some embodiments, the disease comprises a viral infection, a bacterial infection, or a cancer. In some embodiments the condition comprises an IL-2 induced condition. In some embodiments, the IL-2 induced condition comprises pulmonary edema or ar leakage.

The VH and/or VL polypeptides disclosed herein can be administered to a subject (e.g., a human or an animal) alone, or in ation with a carrier, i.e., a pharmaceutically acceptable r. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without g any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. As would be well-known to one of ordinary skill in the art, the carrier is selected to minimize any degradation of the ptides disclosed herein and to minimize any adverse side effects in the subject. The pharmaceutical compositions may be prepared by ology well known in the pharmaceutical art.

The above pharmaceutical compositions comprising the polypeptides disclosed herein can be administered (e. g., to a mammal, a cell, or a tissue) in any suitable manner depending on whether local or ic treatment is desired. For example, the composition can be administered topically (e.g., ophthalmically, vaginally, ly, intranasally, transdermally, and the like), orally, by inhalation, or parenterally ding by intravenous drip or subcutaneous, intracavity, intraperitoneal, intradermal, or intramuscular injection). Topical intranasal administration refers to delivery of the compositions into the nose and nasal passages h one or both of the nares. The composition can be delivered by a spraying mechanism or droplet mechanism, or through aerosolization. Delivery can also be directed to any area of the respiratory system (e.g., lungs) via intubation. Alternatively, stration can be intratumoral, e.g., local or intravenous injection.

If the composition is to be administered parenterally, the administration is generally by injection. ables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for suspension in liquid prior to injection, or as emulsions. Additionally, parental administration can involve preparation of a slow-release or sustained-release system so as to in a constant dosage.

In some embodiments, a composition comprises an anti-IL-2 antibody comprising a heavy chain variable region having the sequence of one of SEQ ID NOs210, l2, l4, l6, 18, 20, 22, 24, 26, or 36. In some embodiments, a ition comprises an anti-IL-2 antibody comprising a light chain variable region having the sequence of one of SEQ ID NOs:ll, 13, 15, l7, 19, 21, 23, 25, 27, or 37.

In some embodiments, a ition comprises an L-2 antibody comprising a heavy chain variable region and a light chain le region having the sequences of one of: SEQ ID NOs: 10 and 11; SEQ ID NOs212 and 13; SEQ ID NOs214 and 15; SEQ ID NOs: 16 and 17; SEQ ID NOs218 and 19; SEQ ID NOs220 and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27, or SEQ ID NOs: 36 and 37. In some ments, a composition comprises an anti-IL-2 antibody comprising a heavy chain variable domain comprising CDRl, CDR2 and CDR3 regions comprising amino acid sequences of SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively; SEQ ID NOs250-52 respectively; SEQ ID -58 respectively; or SEQ ID NOs262-64 respectively. In some embodiments, a composition comprises an anti-IL-2 antibody comprising a light chain variable domain comprising CDRl, CDR2 and CDR3 regions comprising amino acid sequences of SEQ ID NOs241-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-61 respectively; or SEQ ID NOs265-67, respectively. In some embodiments, a composition comprises an anti-IL-2 antibody comprising a heavy chain le domain comprising CDRl, CDR2 and CDR3 s comprising amino acid sequences of SEQ ID NOs238-40 respectively; SEQ ID -46 respectively; SEQ ID NOs250-52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID NOs262-64, tively, and a light chain variable domain comprising CDRl, CDR2, and CDR3 regions comprising amino acid sequences of SEQ ID NOs241-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-61 respectively; or SEQ ID NOs265- 67, respectively.

In some embodiments, a composition comprises an anti-IL-2 antibody sing any of clones BDG 17.014, BDG 17.023, BDG 17.038, BDG 17.043, BDG 17.053, BDG 17.054, BDG 17.066, BDG 17.067, and BDG 17.069. In some embodiments, a composition comprises an anti-IL- 2 antibody comprising anti-IL-2 clone BDG 17.014. In some embodiments, a composition comprises an anti-IL-2 antibody comprising anti-IL-2 clone BDG 17.023. In some embodiments, a ition comprises an L-2 antibody comprising anti-IL-2 clone BDG 17.038. In some ments, a composition comprises an anti-IL-2 antibody comprising L-2 clone BDG 17.043. In some embodiments, a composition comprises an anti-IL-2 dy comprising anti-IL-2 clone BDG 17.053. In some embodiments, a composition comprises an anti-IL-2 antibody comprising anti-IL-2 clone BDG 17.054. In some embodiments, a composition ses an anti-IL-2 antibody comprising anti-IL-2 clone BDG 17.066. In some embodiments, a composition comprises an anti- IL-2 antibody comprising anti-IL-2 clone BDG 17.067. In some embodiments, a composition comprises an anti-IL-2 antibody comprising anti-IL-2 clone BDG 17.069.

In some embodiments, compositions comprise an anti-IL2 antibody and a ceutically acceptable carrier. In some embodiments, compositions comprise an anti-IL2 dy and IL-2, and a ceutically acceptable carrier. In some ments, compositions comprise an anti-IL2 antibody complexed with IL-2, and a pharmaceutically acceptable carrier.

In some embodiments, an anti-IL-2 antibody and IL-2 are comprised in the same composition. In some embodiments, an anti-IL-2 antibody and IL-2 are comprised in different compositions. In some embodiments, administration of a combination of an anti-IL-2 antibody and IL-2, or composition(s) thereof are concurrent. In some embodiments, administration of a ation of an anti-IL-2 antibody and IL-2, or composition(s) thereof comprises administration of an anti-IL-2 dy or a ition thereof, prior to the IL-2 or a composition thereof. In some embodiments, administration of a combination of an anti-IL-2 antibody and IL-2, or composition(s) thereof comprises administration of an anti-IL-2 antibody or a composition thereof, following administration of the IL-2 or a ition thereof.

A skilled artisan would appreciate that a "pharmaceutical composition" may encompass a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active agent, for example but not limited to an antibody or a compound, to an organism.

In some embodiments, sed herein is a ceutical composition for a therapy use treating a subject with a weakened immune system. In some embodiments, disclosed herein is a pharmaceutical composition for a therapy use treating a subject suffering from a viral infection, a ial infection, or a cancer. In some embodiments, sed herein is a pharmaceutical composition for use as part of a combination therapy for treating a subject with a weakened immune system. In some ments, disclosed herein is a pharmaceutical composition for use as part of a ation therapy for use ng a subject suffering from a viral infection, a bacterial infection, or a cancer.

A skilled artisan would appreciate that the phrases "physiologically acceptable carrier", "pharmaceutically able carrier", "physiologically acceptable excipient", and "pharmaceutically acceptable excipient", may be used hangeably may encompass a carrier, excipient, or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered active ingredient.

A skilled artisan would appreciate that an "excipient" may encompass an inert substance added to a pharmaceutical composition to further facilitate administration of an active ient. In some embodiments, excipients include calcium carbonate, calcium ate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs are found in gton’s Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

In some embodiments, the composition as disclosed herein comprises a eutic composition. In some embodiments, the composition as disclosed herein comprises a therapeutic efficacy.

Combination Therapies In some embodiments, an anti-IL-2 antibody or a composition thereof, is used in combination with an immune checkpoint inhibitor. In some ments, the term "immune checkpoint inhibitor" may encompass any compound or molecule capable of inhibiting the function of a checkpoint protein.

In some embodiments, the term "immune checkpoint tor" may encompass any compound or molecule which targets immune checkpoint proteins. An artisan would appreciate that "immune checkpoints" are key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Checkpoint inhibitors can block inhibitory checkpoints, and thereby restore immune system function. In some ments, the one or more checkpoint inhibitors comprise immune checkpoint inhibitors.

A skilled artisan would iate that the terms e checkpoint tors" (ICIs), "checkpoint inhibitors," and the like may be used interchangeably herein having all the same qualities and meanings, wherein an immune checkpoint inhibitor encompasses compounds that inhibit the activity or control mechanism(s) of the immune . Immune system checkpoints, or immune checkpoints, are inhibitory pathways in the immune system that generally act to maintain self- tolerance or te the duration and amplitude of logical immune ses to minimize collateral tissue damage. Checkpoint inhibitors can inhibit an immune system checkpoint by inhibiting the activity of a protein in the y.

Immune checkpoint inhibitor targets include, but are not limited to PD-l, PDL-l, CTLA-4, TIGIT, TIM—3, B7—H3, CD73, LAG3, CD27, CD70, 4—1BB, GITR, 0X40, SIRP—alpha (CD47), CD39, ILDR2, VISTA, BTLA, and VTCN—l. In some embodiments, an anti-IL-2 antibody therapy is used in combination With an immune checkpoint tor, wherein the target of the immune checkpoint inhibitor comprises PD- 1, PDL- 1, , TIGIT, TIM-3, B7-H3, CD73, LAG3, CD27, CD70, 4—1BB, GITR, 0X40, SIRP—alpha (CD47), CD39, ILDR2, VISTA, BTLA, or VTCN—l, or any combination thereof.

Checkpoint inhibitors may include antibodies, or antigen binding nts thereof, other binding proteins, biologic therapeutics, or small molecules, that bind to and block or inhibit the actiVity of one or more of PD-1, PDL-l, CTLA-4, TIGIT, TIM-3, B7-H3, CD73, LAG3, CD27, CD70, 4—1BB, GITR, 0X40, SIRP—alpha (CD47), CD39, ILDR2, VISTA, BTLA, or VTCN—l.

Illustrative checkpoint inhibitors include but are not limited to those listed in Table 1 below. id="p-129" id="p-129" id="p-129"

[0129] Table 1: Non-Limiting Examples of Checkpoint Inhibitors and the Immune oint Inhibitor Target.

Cemiplimab Camrelizumab COM—902 anti—TIGIT Sabatolimab anti-TIM3 INCAGN—1949 anti-0X40 (agonist) 74998 anti-0X40 (agonist) BGB-A-445 anti-0X40 (agonist) In some embodiments, the checkpoint inhibitor comprises a PD-1 inhibitor. In some embodiments, the checkpoint inhibitor comprises a PDL-1 inhibitor. In some embodiments, the checkpoint inhibitor comprises a CTLA-4 tor. In some ments, the checkpoint inhibitor ses a TIGIT inhibitor. In some embodiments, the checkpoint inhibitor comprises a TIM-3 tor. In some embodiments, the checkpoint inhibitor comprises a B7-H3 inhibitor. In some embodiments, the oint inhibitor comprises a CD73 inhibitor. In some embodiments, the checkpoint inhibitor comprises a LAG3 inhibitor. In some ments, the checkpoint inhibitor comprises a CD27 inhibitor. In some embodiments, the checkpoint inhibitor comprises a CD70 inhibitor. In some embodiments, the checkpoint tor comprises a 4-1BB agonist binder. In some embodiments, the checkpoint inhibitor comprises a GITR agonist binder. In some embodiments, the checkpoint inhibitor comprises a 0X40 agonist binder. In some embodiments, the checkpoint tor comprises a SIRP-alpha (CD47) inhibitor. In some embodiments, the checkpoint inhibitor comprises a CD39 inhibitor. In some embodiments, the checkpoint inhibitor comprises a ILDR2 inhibitor. In some embodiments, the checkpoint inhibitor comprises a VISTA tor. In some embodiments, the checkpoint inhibitor comprises a BTLA tor. In some embodiments, the checkpoint inhibitor comprises a VTCN—l inhibitor.

In some embodiments, the checkpoint inhibitor comprises a combination of a PD-l inhibitor, a PDL-l inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, a CD73 inhibitor, a LAG3 inhibitor, a CD27 inhibitor, a CD70 inhibitor, a 4-1BB inhibitor, a GITR inhibitor, a 0X40 inhibitor, a SIRP-alpha (CD47) inhibitor, a CD39 inhibitor, a ILDR2 inhibitor, a VISTA inhibitor, a BTLA inhibitor, a VTCN—l inhibitor. In some embodiments, the checkpoint inhibitor comprises at least two checkpoint tors selected from of a PD-l inhibitor, a PDL-l inhibitor, a CTLA-4 tor, a TIGIT inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, a CD73 inhibitor, a LAG3 inhibitor, a CD27 inhibitor, a CD70 inhibitor, a 4-1BB inhibitor, a GITR inhibitor, a 0X40 inhibitor, a lpha (CD47) inhibitor, a CD39 inhibitor, a ILDR2 inhibitor, a VISTA inhibitor, a BTLA inhibitor, and a VTCN—l inhibitor.

In some embodiments, a pharmaceutical composition for use in a combination therapy, as described herein, comprises an effective amount of a checkpoint inhibitor, as described herein, and a pharmaceutically acceptable carrier.

In some ments, a ition disclosed herein comprises a oint inhibitor and a ceutically acceptable carrier. In some embodiments, a composition disclosed herein comprises a combination of checkpoint inhibitors, and a pharmaceutically acceptable carrier. In some embodiments, a composition comprises a checkpoint inhibitor comprising a PD-l inhibitor, a PDL-l tor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM-3 tor, a B7-H3 inhibitor, a CD73 inhibitor, a LAG3 inhibitor, a CD27 tor, a CD70 inhibitor, a 4-1BB tor, a GITR inhibitor, a 0X40 inhibitor, a SIRP-alpha (CD47) inhibitor, a CD39 inhibitor, a ILDR2 inhibitor, a VISTA inhibitor, a BTLA inhibitor, a VTCN—l inhibitor. In some embodiments, the checkpoint inhibitor comprises at least two checkpoint inhibitors ed from of a PD-l inhibitor, a PDL-l inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM-3 inhibitor, a B7-H3 tor, a CD73 inhibitor, a LAG3 inhibitor, a CD27 inhibitor, a CD70 inhibitor, a 4-1BB inhibitor, a GITR inhibitor, a 0X40 inhibitor, a SIRP-alpha (CD47) inhibitor, a CD39 inhibitor, a ILDR2 inhibitor, a VISTA inhibitor, a BTLA inhibitor, or a VTCN—l inhibitor, and a pharmaceutically acceptable r. In some embodiments, a composition comprises at least two checkpoint tors selected from a PD-l inhibitor, a PDL-l inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, a CD73 inhibitor, a LAG3 tor, a CD27 inhibitor, a CD70 inhibitor, a 4-1BB inhibitor, a GITR inhibitor, a 0X40 inhibitor, a lpha (CD47) inhibitor, a CD39 inhibitor, a ILDR2 inhibitor, a VISTA inhibitor, a BTLA tor, a VTCN—l inhibitor. In some embodiments, the checkpoint inhibitor comprises at least two checkpoint inhibitors selected from of a PD-l inhibitor, a PDL-l inhibitor, a CTLA-4 inhibitor, a TIGIT tor, a TIM-3 inhibitor, a B7-H3 inhibitor, a CD73 inhibitor, a LAG3 inhibitor, a CD27 tor, a CD70 inhibitor, a 4-1BB inhibitor, a GITR inhibitor, a 0X40 inhibitor, a SIRP-alpha (CD47) inhibitor, a CD39 inhibitor, a ILDR2 inhibitor, a VISTA inhibitor, a BTLA inhibitor, and a VTCN—l inhibitor, and a pharmaceutically acceptable carrier.

In certain embodiments, when more than one checkpoint inhibitor is used in a therapeutic method bed herein, each oint inhibitor is comprised within a separate composition. In certain embodiments, when more than one checkpoint inhibitor is used in a therapeutic method described herein, oint inhibitor may be comprised within the same composition.

In some embodiments, a ation y ses use of an anti-IL-2 antibody or composition thereof as described , and a checkpoint inhibitor or a composition thereof. In some embodiments, a combination therapy comprises use of an anti-IL-2 antibody or composition thereof and IL-2 as described herein, and a checkpoint inhibitor or a composition thereof. In some embodiments, a combination therapy comprises use of an anti-IL-2 dy or composition thereof complexed with IL-2 as described herein, and a checkpoint inhibitor or a composition thereof.

In some ments, a combination therapy comprises use of an anti-IL-2 antibody or composition thereof as described herein, and at least two checkpoint tors or a composition thereof. In some embodiments, a combination therapy comprises use of an anti-IL-2 antibody or composition thereof and IL-2 as described herein, and at least two checkpoint inhibitors or a composition thereof. In some embodiments, a combination therapy comprises use of an anti-IL-2 antibody or composition thereof complexed with IL-2 as described herein, and at least two checkpoint inhibitors or a composition thereof.

In some embodiments, a combination therapy comprises a second composition comprising one or more oint inhibitors, as described herein.

In some embodiments of a combination therapy, an anti-IL-2 antibody and IL-2 are sed in the same composition as a checkpoint inhibitor. In some embodiments, an anti-IL-2 antibody and IL-2 are sed in different compositions from each other and from a checkpoint inhibitor.

In some embodiments of a combination therapy, the order of administration of an anti-IL-2 antibody or a composition thereof and a checkpoint inhibitor or a composition thereof, may be in any order. In some embodiments of a combination therapy, the order of administration of an anti-IL-2 antibody or a composition f, IL-2 or a composition thereof, and a checkpoint tor or a composition thereof, may be in any order. For example, but not limited to the anti-IL-2 dy may be administered prior to, concurrent with, or following administration of the checkpoint inhibitor.

Similarly, a combination of an anti-IL-2 antibody and IL-2 may be administered prior to, concurrent with, or following administration of the checkpoint inhibitor. In some embodiments, the anti-IL-2 antibody may be administered prior to, concurrent with, or following administration of the at least two checkpoint inhibitors. rly, a combination of an anti-IL-2 antibody and IL-2 may be administered prior to, concurrent with, or following administration of the at least two checkpoint inhibitors.

In some embodiments, administration of a ation therapy with a checkpoint inhibitor comprises concurrent administration of an anti-IL-2 antibody or a ition thereof and the checkpoint inhibitor. In some embodiments, administration of a combination therapy with a checkpoint inhibitor comprises concurrent administration of an L-2 antibody and IL-2, or composition(s) thereof and the oint inhibitor. In some embodiments, administration of a combination therapy with a checkpoint inhibitor comprises prior stration of an anti-IL-2 antibody or a composition thereof before the checkpoint inhibitor. In some embodiments, administration of a combination therapy with a checkpoint inhibitor comprises prior administration of an anti-IL-2 dy and IL-2, or composition(s) thereof before the checkpoint inhibitor. In some embodiments, administration of a combination therapy with a checkpoint inhibitor comprises later administration of an anti-IL-2 antibody or a composition thereof following administration of the checkpoint inhibitor. In some embodiments, stration of a combination y with a checkpoint inhibitor comprises later administration of an anti-IL-2 antibody and IL-2, or composition(s) thereof following the administration of the checkpoint inhibitor.

In some embodiments, a ation therapy comprises use of a checkpoint inhibitor and an anti-IL-2 antibody comprising a heavy chain variable region having the sequence of one of SEQ ID NOs: 10, l2, l4, 16, 18, 20, 22, 24, 26, or 36. In some embodiments, a combination therapy comprises use of a checkpoint tor and an L-2 antibody comprising a light chain variable region having the sequence of one of SEQ ID NOs21 l, 13, 15, l7, 19, 21, 23, 25, 27, or 37. In some embodiments, a ation therapy comprises use of a checkpoint inhibitor and an anti-IL-2 antibody comprising a heavy chain variable region and a light chain variable region having the sequences of one of: SEQ ID NOs210 and 11; SEQ ID NOs212 and 13; SEQ ID NOs214 and 15; SEQ ID NOs216 and 17; SEQ ID NOs218 and 19; SEQ ID NOs220 and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27, or SEQ ID NOs: 36 and 37. In some embodiments, a combination therapy comprises use of a checkpoint inhibitor and an anti-IL-2 antibody comprising a heavy chain variable domain comprising CDRl, CDR2 and CDR3 regions comprising amino acid sequences of SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 tively; SEQ ID NOs250-52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID -64 respectively. In some embodiments, a combination therapy comprises use of a checkpoint inhibitor and an anti-IL-2 dy comprising a light chain variable domain comprising CDRl, CDR2 and CDR3 regions comprising amino acid sequences of of SEQ ID NOs241-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253—55 respectively; SEQ ID NOs259-61 respectively; or SEQ ID NOs265-67, respectively. In some ments, a combination y ses use of a checkpoint inhibitor and an anti-IL-2 antibody comprising a heavy chain variable domain sing CDRl, CDR2 and CDR3 regions comprising amino acid sequences of SEQ ID -40 respectively; SEQ ID NOs244-46 respectively; SEQ ID NOs250-52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID NOs262- 64, respectively, and a light chain variable domain comprising CDRl, CDR2, and CDR3 regions comprising amino acid sequences of SEQ ID NOs241-43 tively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-61 respectively; or SEQ ID NOs265- 67, respectively.

In some embodiments, a combination therapy comprises use of a checkpoint inhibitor and an anti-IL-2 antibody comprising any of clones BDG 17.014, BDG 17.023, BDG 17.038, BDG 17.043, BDG 17.053, BDG 17.054, BDG 17.066, BDG 17.067, and BDG 17.069. In some embodiments, a combination therapy ses use of a checkpoint inhibitor and an anti-IL-2 antibody comprising L-2 clone BDG 17.014. In some embodiments, a combination therapy comprises use of a checkpoint inhibitor and an anti-IL-2 antibody comprising anti-IL-2 clone BDG 17.023. In some embodiments, a combination therapy comprises use of a checkpoint inhibitor and an anti-IL-2 antibody comprising anti-IL-2 clone BDG 17.038. In some embodiments, a combination therapy comprises use of a oint inhibitor and an anti-IL-2 antibody comprising anti-IL-2 clone BDG 17.043. In some embodiments, a combination therapy comprises use of a checkpoint inhibitor and an anti-IL-2 dy comprising L-2 clone BDG 17.053. In some ments, a combination therapy comprises use of a checkpoint inhibitor and an anti-IL-2 dy comprising anti-IL-2 clone BDG 17.054. In some embodiments, a combination therapy comprises use of a checkpoint inhibitor and an anti-IL-2 antibody comprising anti-IL-2 clone BDG 17.066. In some embodiments, a combination therapy comprises use of a checkpoint inhibitor and an anti-IL-2 antibody sing anti-IL-2 clone BDG 17.067. In some embodiments, a combination therapy comprises use of a checkpoint tor and an anti-IL-2 antibody comprising anti-IL-2 clone BDG 17.069.

Formulations Pharmaceutical compositions sed herein comprising anti-IL-2 dies, or a combination of L-2 antibodies and IL-2, or checkpoint inhibitors, can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, Which may be buffered to a selected pH, Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more ient to administer, especially by injection. Viscous itions, on the other hand, can be formulated Within the riate viscosity range to e longer contact periods With specific tissues. Liquid or viscous compositions can comprise rs, Which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for e, glycerol, ene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof. e injectable solutions can be prepared by incorporating the anti-IL-2 antibodies, or a combination of anti-IL-2 antibodies and IL-2, or checkpoint inhibitors, described herein and utilized in practicing the methods disclosed herein, in the required amount of the appropriate t With s amounts of the other ingredients, as desired. Such formulations may be in admixture With a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The formulations can also be lyophilized. The formulations can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e. g., methylcellulose), pH buffering agents, gelling or ity enhancing ves, preservatives, ?avoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON‘S PHARMACEUTICAL E", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare le preparations, Without undue experimentation. id="p-145" id="p-145" id="p-145"

[0145] Various additives Which enhance the stability and sterility of the formulations, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal , for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. ged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

In certain embodiments, the terms "pharmaceutical composition", sition", and "formulation" may be used interchangeably having the same meanings and qualities.

The compositions or ations described herein can be isotonic, i.e., they can have the same osmotic re as blood and lacrimal ?uid. The desired isotonicity of the compositions as disclosed herein may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, ene glycol or other nic or c solutes. Sodium chloride may be preferred particularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose may be preferred because it is readily and economically available and is easy to work with. id="p-149" id="p-149" id="p-149"

[0149] Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of le carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e. g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

In some embodiments, a composition is formulated to be at a pH between about pH 5.0 - 6.0.

In some embodiments, a ition is formulated to be at a pH between about pH 5.0 - 7.0. In some embodiments, a composition is formulated to be at a pH between about pH 5.0 - 6.5. In some embodiments, a composition is ated to be at a pH between about pH 5.0 — 5.5. In some embodiments, a composition is formulated to be at a pH between about pH 5.5 - 6.0. In some ments, a composition is formulated to be at a pH n about pH 5.5 - 6.5. In some embodiments, a composition is ated to be at a pH between about pH 5.0. In some embodiments, a composition is formulated to be at a pH between about pH 5.5. In some embodiments, a composition is formulated to be at a pH between about pH 6.0. In some embodiments, a composition is formulated to be at a pH between about pH 6.5 In some embodiments, a ition is formulated to be at a pH between about pH 5.0 - 6.0 and comprises a buffer. In seme embodiments, the buffer comprises a pharmaceutically acceptable buffer. In some embodiments, the buffer comprises a histidine buffer or a citrate buffer. In some ments, the buffer comprises a histidine buffer. In some embodiments, the buffer comprises a citrate buffer. I In some ments, a composition is formulated to be at a pH between about pH 5.0 - 6.0 and comprises a buffer ed from a histidine buffer and a citrate buffer. In some embodiments, a composition is formulated to be at a pH between about pH 5.0 - 6.0 and comprises a histidine buffer.

In some embodiments, a composition is formulated to be at a pH between about pH 5.0 - 6.0 and comprises a citrate buffer.

In some embodiments, a composition further comprises at least one of sucrose, methionine, or P380, or any combination thereof. In some embodiments, a composition further comprises sucrose.

In some embodiments, a composition further comprises methionine. In some embodiments, a composition r ses P380. id="p-154" id="p-154" id="p-154"

[0154] In some embodiments, a ition comprises an anti-IL-2 antibody as disclosed herein and is formulated to be at a pH between about pH 5.0 - 6.0 and comprises a buffer selected from a histidine buffer and a citrate buffer. In some embodiments, the composition further comprises IL-2.

Those skilled in the art will ize that the components of the compositions or formulations should be selected to be chemically inert and will not affect the viability or efficacy of the early tic cell populations as bed herein, for use in the methods disclosed herein. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

Methods of Use id="p-156" id="p-156" id="p-156"

[0156] In one embodiment, the present disclosure provides a method of producing a heavy chain variable region of an anti-IL-2 antibody, the method comprises the step of culturing host cells under conditions conducive to expressing a vector encoding for the heavy chain variable region, y producing the heavy chain le region of the anti-IL—2 antibody.

In one embodiment, the present disclosure es a method of producing a light chain variable region of an anti-IL-2 dy, the method comprises the step of culturing host cells under conditions conducive to expressing a vector encoding for the light chain variable region, thereby producing the light chain variable region of the anti-IL-2 antibody.

The VH and/or VL polypeptides disclosed herein may be used in eutic methods. In one embodiment, the polypeptides of the present disclosure can be used as immunotherapeutic agents, for example, for differential activation of immune cells as described herein. The present polypeptides can be administered to a t ly, or by administering to the subject a nucleic acid sequence encoding the polypeptides, such nucleic acid sequence may be d by a vector.

The exact amount of the present polypeptides or compositions thereof required to elicit the desired s will vary from subject to subject, depending on the species, age, gender, weight, and general ion of the subject, the particular polypeptides, the route of administration, and whether other drugs are included in the regimen. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using routine experimentation. Dosages can vary, and the polypeptides can be administered in one or more (e.g., two or more, three or more, four or more, or five or more) doses daily, for one or more days. Guidance in selecting appropriate doses for dies can be y found in the literature. id="p-160" id="p-160" id="p-160"

[0160] In some ments of a methods of use of an anti-IL-2 antibody described herein, a subject comprises a mammalian subject. In some embodiments, a subject comprises a human subject. In some embodiments, a subject suffers from immune deficiency problems. Treatment of an immune deficient subject would in some embodiments, comprise a prophylactic treatment.

In one ment, the present disclosure provides a method ofpromoting differential growth of immune cells in a subject, comprising the step of preparing a composition comprising an anti-IL- 2 dy disclosed herein, and administering the ition to the subject, thereby promoting differential growth of immune cells in the subject. In one ment, the present disclosure es a method of promoting differential growth of immune cells in a subject, comprising the step of preparing a composition comprising IL-2 and the anti-IL-2 dy disclosed herein, and administering the composition to the subject, thereby promoting differential growth of immune cells in the subject. In one embodiment, the subject can be an animal or a human. In one embodiment, the immune cells can be CD8+ cells or NK cells.

In some embodiments, disclosed herein is a method of treating a disease or a condition in a subject, comprising the step of administering to the subject a composition comprising an anti-IL-2 dy as disclosed herein, wherein said antibody promotes differential growth of subsets of immune cells and decreases undesirable effects caused by IL-2, thereby treating said disease or condition in said subject. In some embodiments, a method of treating a disease sed here comprises use of a composition comprising an anti-IL-2 antibody and IL-2, or ananti-IL-2 antibody complexed with IL-2. In some embodiments, a method of treating a disease ses treating a viral infection, a bacterial ion, or a cancer. In some embodiments, a method of treating a condition comprises treating a weak immune system and the treatment prophylactically boosts the immune system.

In some embodiments of a method of treating a disease or a condition, the condition comprises a genetic predisposition that increases likelihood of cancer in said subject. In some embodiments, the genetic predisposition comprises a change in expression or activity of a gene product. In some embodiments, a genetic predisposition that ses the likelihood of cancer comprises a mutation in a tumor suppressor gene or a mismatch repair (MMR) gene, or a combination thereof.

Many hereditary cancers are known in the art non-limiting examples include but are not limited to Hereditary Breast and Ovarian Cancer (HB OC) me, Lynch syndrome (hereditary lyposis colorectal cancer), and Li-Fraumeni syndrome. id="p-165" id="p-165" id="p-165"

[0165] In some embodiments, the genetic predisposition increases the likelihood of HBOC. HBOC is associated with mutations in the BRACl and BRAC2 genes. HBOC is associated with a number of different s not just breast cancer, including but not limited to fallopian tube cancer, primary peritoneal cancer, male breast , pancreatic , and prostate cancer. In some embodiments, the genetic position ses the likelihood of any of breast cancer, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, male breast cancer, pancreatic cancer, or prostate cancer, or a combination thereof.

In some embodiments, the c predisposition ses the likelihood of hereditary non- polyposis colorectal cancer (HNPCC). HNPCC is ated with mutation in genes including but not d to MLHl, MSHZ, MSH6, PMSl, and PMSZ. HNPCC is associated with high risk of developing endometrial cancer, as well as s of the ovary, stomach, small intestine, pancreas, kidney, brain, ureters, and bile duct. In some embodiments, the genetic predisposition increases the likelihood of any of hereditary non-polyposis colorectal cancer, cancer of the ovary, stomach cancer, cancer of the small intestine, pancreatic cancer, kidney cancer, brain cancer, cancer of ureters, and cancer of a bile duct. id="p-167" id="p-167" id="p-167"

[0167] In some embodiments, the genetic predisposition increases the likelihood of Li-Fraumeni syndrome. Li-Fraumeni syndrome in genes including but not limited to TP53 and CHEKZ, or a combination thereof. Li-Fraumeni syndrome is associated with cancers, including sarcoma, osteosarcoma, issue sarcomas, leukemia, brain (central nervous system) cancers, cancer of the adrenal cortex and breast cancer, or ations thereof. In some embodiments, the genetic predisposition increases the likelihood of any of a sarcoma, osteosarcoma, soft-tissue sarcomas, leukemia, brain (central nervous system) cancers, cancer of the adrenal cortex and breast cancer, or combinations thereof.

In some embodiments, a condition being treated in a subject, comprises treating a subject with a genetic predisposition comprising a change in expression or activity of a gene t, said gene comprising BRCAl, BRAC2, MLHl, MSH2, MSH6, PMSl, PMS2, TP53, or CHEK2, or a combination f.

As described herein, a complex of IL-2 and the anti-IL-2 antibodies sed exhibited pronounced effect in inducing eration of memory phenotype effector T cells (MP) CD8+ cells and NK cells, while there was much smaller effect on CD4+ Tregs. Thus, the engineered anti-IL-2 antibodies disclosed herein would be useful in adjusting immune cell populations and ng differential expansion of certain immune or cells. In one embodiment, such differential ion of immune effect cells would result in robust activation of the immune system and could be useful for treatment of tumors. In some embodiments, treatment comprising treating a solid tumor.

In some embodiments, treatment comprises treating a non-solid tumor. In some embodiments, treating comprising treating solid and or non-solid tumors, such as but not d to melanoma, renal cell oma, small cell lung cancer or other cancer conditions. In another embodiment, the method disclosed herein would be useful for treatment of viral infection or bacterial infection. In another embodiment, the method disclosed herein would be useful for ng or preventing a condition caused by IL-2 binding to elial CD25 expressing cells, e. g., pulmonary edema, or ILinduced vascular leakage. id="p-170" id="p-170" id="p-170"

[0170] In some embodiments of a methods of treating a disease or condition, the immune cells showing differential growth comprise one or more of naive T cells, memory T cells, CD8+ T cells, NK cells, or Natural Killer T cells. In some embodiments of a methods of treating a disease or condition, the undesirable effect caused by IL-2 comprises one or more of tion of tory T cells, apoptosis of CD25+ T effector cells, IL-2 induced pulmonary edema, IL-2 induced pneumonia, or ILinduced vascular leakage. In some embodiments of a method of ng a disease or condition, an anti-IL-2 antibody disclosed herein inhibits IL-2 binding to CD25.

In some embodiments, treatment of cancer comprises maintenance treatments. In some embodiments, maintenance treatments are administered to in the absence of a cancer or tumor.

In some embodiments, maintenance treatments are administered to maintain lack of metastasis of a cancer or tumor. In some embodiments, nance treatments are administered to inhibit metastasis of a cancer or tumor. In some embodiments, maintenance treatments are stered to maintain lack of growth of a cancer or tumor. In some embodiments, maintenance treatments are administered to inhibit growth of a cancer or tumor.

In some embodiments, treatment of cancer comprises prophylactic ent, for example but not limited to a subject ing a genetic marker or markers with a high risk of developing cancer.

In some embodiments, the genetic marker ses a mutation in the BRCA1 gene.

In some embodiments of methods of promoting ential growth of immune cells in a t, comprising the step of preparing and administering a composition comprising an anti-IL-2 antibody disclosed herein.

In some embodiments of methods of promoting differential growth of immune cells in a subject, comprising the step of preparing and administering a composition comprising IL-2 and an anti-IL-2 antibody disclosed herein, the administration of a combination of an anti-IL-2 antibody and IL-2, or composition(s) thereof are concurrent. In some embodiments of methods of ing differential growth of immune cells in a t, comprising the step of preparing and administering a composition comprising IL-2 and an L-2 antibody, the administration of an anti-IL-2 antibody and IL-2, or composition(s) thereof comprises administration of an anti-IL-2 dy or a composition thereof, prior to the IL-2 or a composition thereof. In some embodiments of methods of promoting differential growth of immune cells in a subject, sing the step of ing and administering a composition comprising IL-2 and an anti-IL-2 antibody, the administration of an anti- IL-2 antibody and IL-2, or composition(s) thereof comprises administration of an anti-IL-2 antibody or a composition thereof, following administration of the IL-2 or a composition thereof.

In some embodiments, the present disclosure provides a method of treating a subject with a disease or a condition through induction of ential growth of immune cells. In one embodiment, the disease can be viral infection, bacterial infection, or cancer. In one embodiment, the condition can be IL-2 induced ary edema, or ILinduced vascular leakage. The method comprises the step of: (a) preparing a composition comprising an anti-IL-2 antibody as disclosed herein; and (b) administering the composition from (a) to the subject, thereby treating the subject through differential growth of immune cells in the subject. In certain embodiments, any of the engineered anti-IL-2 dies disclosed herein may be used in the method of ent as described.

In some embodiments, the present disclosure provides a method of treating a subject with a disease or a condition through induction of differential growth of immune cells. In one embodiment, the disease can be viral infection, ial infection, or . In one embodiment, the condition can be IL-2 induced pulmonary edema, or ILinduced ar leakage. The method comprises the step (a) preparing a composition comprising IL-2 and an anti-IL-2 antibody as disclosed ; and (b) administering the composition from (a) to the t, y ng the t through differential growth of immune cells in the subject. In addition to facilitate expansion of subsets of immune effector cells, the antibodyHL-Z complex would also decrease undesirable effects caused by IL-2 (e.g., IL-2 induced pulmonary edema, or ILinduced vascular leakage). In one embodiment, the subject can be an animal or a human. In certain ments, any of the engineered anti-IL-2 antibodies disclosed herein may be used in the method of treatment as described.

In one embodiment, the present disclosure provides a method of treating a disease or a condition in a subject (e.g., an animal or a human), comprising the step of administering to the subject a composition comprising anti-IL-2 antibodies, wherein the antibodies facilitate expansion of subsets of immune cells and decrease undesirable effects caused by IL-2, thereby treating the disease or condition in the subject. In some embodiments of a method of treating a disease or a condition in a subject, comprising the step of preparing and administering a composition comprising an anti-IL-2 antibody disclosed herein, and administering the composition comprising the anti-IL-2 antibody. In one ment, the composition comprises IL-2 and the L-2 antibodies as disclosed herein, or the ition comprises anti-IL-2 antibodies that are complexed with IL-2.

In some embodiments of a method of treating a disease or a condition in a subject, comprising the step of preparing and administering a composition comprising IL-2 and an L-2 antibody sed herein, the administration of a combination of an anti-IL-2 antibody and IL-2, or composition(s) thereof are concurrent. In some embodiments of a method of treating a e or a condition in a subject, comprising the step of preparing and administering a composition comprising IL-2 and an anti-IL-2 dy, the administration of an anti-IL-2 antibody and IL-2, or composition(s) thereof comprises administration of an anti-IL-2 antibody or a composition thereof, prior to the IL-2 or a composition thereof. In some embodiments of a method of treating a disease or a condition in a subject, comprising the step aring and administering a composition comprising IL-2 and an anti-IL-2 antibody, the administration of an anti-IL-2 antibody and IL-2, or composition(s) thereof comprises administration of an L-2 antibody or a composition thereof, following stration of the IL-2 or a composition thereof.

In one embodiment, the method of treatment would be effective for treating condition such as IL-2 induced pulmonary edema, or ILinduced ar leakage. In another embodiment, the method of treatment would be effective for treating ary edema (mild or chronic) resulted from viral or bacterial infections.

In one embodiment, the disease can be viral infection, bacterial infection, cancer, mune disease or immune disorder. In one embodiment, the e can be upper respiratory viral ions, early-stage lung ions, or late stage lung infections. A number of diseases and cancer are known to be caused by s. Examples of disease-causing viruses include, but are not limited to, norovirus; rotavirus; hepatitis virus A, B, C, D, or E; rabies virus, West Nile virus, enterovirus, echovirus, coxsackievirus, herpes simplex virus (HSV), HSV-2, varicella-zoster virus, mosquito- bome viruses, arbovirus, St. Louis encephalitis virus, California encephalitis virus, lymphocytic choriomeningitis virus, human deficiency virus (HIV), poliovirus, zika virus, rubella virus, cytomegalovirus, human papillomavirus (HPV), enterovirus D68, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome coronavirus, SARS coronavirus 2, Epstein- Barr virus, in?uenza virus, respiratory syncytial virus, polyoma viruses (such as JC virus, BK virus), Ebola virus, Dengue virus, or any combination thereof. In one embodiment, the viral infection is caused by SARS CoV-2. In another embodiment, the cancer can be, but is not limited to, melanoma or renal cell oma. id="p-181" id="p-181" id="p-181"

[0181] In one embodiment, the immune cells that are expanded by treatment with the anti-IL-2 antibodies se one or more of naive T cells, memory T cells, CD8+ T cells, NK cells, and Natural Killer T cells. In one embodiment, treatment with the L-2 dies would decrease one or more undesirable effects caused by IL-2 such as activation of regulatory T cells, apoptosis of CD25+ T effector cells, pulmonary edema, pneumonia, and ILinduced vascular leakage. id="p-182" id="p-182" id="p-182"

[0182] In one embodiment, the anti-IL-2 antibodies administered in the above method are engineered or modified anti-IL-2 antibodies that can inhibit IL-2 binding to CD25. In some embodiments, the engineered or modified anti-IL-2 antibodies comprise a heavy chain variable region having the sequence of one of SEQ ID NOs: 10, 12, 14, l6, 18, 20, 22, 24, 26, or 36. In some ments, the engineered or modified anti-IL-2 antibodies se a light chain variable region having the sequence of one of SEQ ID NOs21 l, 13, 15, l7, 19, 21, 23, 25, 27, or 37. In some embodiments, the engineered or modified anti-IL-2 antibodies comprise a heavy chain variable region and a light chain variable region having the sequences of one of: SEQ ID NOs210 and 11; SEQ ID NOs212 and 13; SEQ ID NOs:l4 and 15; SEQ ID NOs:l6 and 17; SEQ ID NOs:l8 and 19; SEQ ID NOs220 and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27; or SEQ ID NOs: 36 and id="p-183" id="p-183" id="p-183"

[0183] In another embodiment, the engineered or modified anti-IL-2 antibodies comprise a heavy chain variable region having complementarity determining region (CDR) l, CDR2 and CDR3. In one ment, the heavy chain CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively; SEQ ID NOs250-52 respectively; SEQ ID NOs256—58 tively; or SEQ ID NOs262-64 respectively. id="p-184" id="p-184" id="p-184"

[0184] In another embodiment, the engineered or ed anti-IL-2 antibodies comprise a light chain variable region having complementarity determining region (CDR) l, CDR2 and CDR3. In one embodiment, the light chain CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs24l-43 tively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID NOs265-67 tively. id="p-185" id="p-185" id="p-185"

[0185] In some embodiments, the engineered anti-IL-2 antibody can be an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, or a F(ab’)2. The IgG can be of the subclass of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the ered antibody can be part of a minibody, a diabody, a triabody, a nanobody, or a single domain antibody.

In some embodiments, a polynucleotide sequence encoding an engineered anti-IL-2 antibody is used in a method of treating a subject With a disease or condition as described herein, n the polynucleotide encodes an antibody comprising a heavy chain le region having the amino acid sequence of one of SEQ ID , l2, l4, l6, 18, 20, 22, 24, 26, or 36 In some embodiments, a polynucleotide sequence encoding an engineered anti-IL—2 dy is used in a method of treating a subject With a disease or condition as described herein, wherein the polynucleotide encodes an antibody comprising a light chain variable region having the amino acid ce of one of SEQ ID NOs:ll, 13, 15, l7, 19, 21, 23, 25, 27, or 37. In some embodiments, a polynucleotide sequence encoding an engineered anti-IL-2 antibody is used in a method of treating a subject with a disease or condition as described herein, wherein the polynucleotide encodes an antibody comprising a heavy chain variable region and a light chain variable region having the amino acid sequences of one of: SEQ ID NOs210 and 11; SEQ ID NOs:l2 and 13; SEQ ID NOs214 and 15; SEQ ID NOs: l6 and 17; SEQ ID NOs: l8 and 19; SEQ ID NOs220 and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27; or SEQ ID NOs: 36 and 37.

In some embodiments of a method of using a polynucleotide to treat a disease or ion as described above, the polynucleotide encodes an engineered anti-IL-2 antibody that can be an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, or a F(ab’)2. The IgG can be of the subclass of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the polynucleotide encodes an engineered antibody which is part of a minibody, a diabody, a dy, a nanobody, or a single domain antibody.

In some embodiments a polynucleotide sequence ng an engineered anti-IL-2 antibody is used in a method of treating a t with a disease or condition as described herein, wherein the polynucleotide sequence comprises the sequence of one of SEQ ID NOS: 1, 2, 3, 4, 5, 31, 32, 33, 34, or 35.

In some embodiments of a method of treating a disease or condition as described herein, the immune effector cells that are activated by the treatment are CD8+ cells or NK cells. In one embodiment, the anti-IL-2 dies disclosed herein, or a complex of IL-2 and the anti-IL-2 dies disclosed , exhibits pronounced effect in ng proliferation of MP CD8+ cells and NK cells, while there was much smaller effect on CD4+ Tregs. In certain embodiments, there is no effect on CD4+Tregs.

In certain embodiments, methods of use of an L-2 antibody disclosed herein provide a imulatory effect. A skilled artisan would appreciate that use of the anti-IL-2 antibodies described and exemplified herein e.g., Example 1, clearly demonstrate a pro-stimulatory effect as opposed to an anti-stimulatory or pro-regulatory .

In some embodiments, use of an engineered or modified anti-IL-2 antibody comprising a heavy chain variable region having the sequence of one of SEQ ID NOs210, l2, l4, l6, 18, 20, 22, 24, 26, or 36, provides a pro-stimulatory immune effect in a subject in need thereof. In some embodiments, use of an engineered or ed anti-IL-2 antibody comprising a light chain variable region having the sequence of one of SEQ ID NOs:ll, 13, 15, l7, 19, 21, 23, 25, 27, or 37, provides a pro-stimulatory immune effect in a subject in need thereof. In some embodiments, use of an engineered or modified anti-IL-2 antibody comprising a heavy chain variable region and a light chain variable region having the sequences of one of: SEQ ID NOs210 and 11; SEQ ID NOs212 and 13; SEQ ID NOs214 and 15; SEQ ID NOs216 and 17; SEQ ID NOs218 and 19; SEQ ID NOs22O and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27; or SEQ ID NOs: 36 and 37, provides a pro-stimulatory immune effect in a subject in need f. In some embodiments, said use comprises the anti-IL-2 antibody. In some embodiments, said use comprises the anti-IL-2 antibody and an IL-2. In some embodiments, use comprises a complex of an anti-IL-2 antibody With an IL-2.

In some embodiments, use of an engineered or modified anti-IL-2 antibody comprising a heavy chain le region having the sequence of one of SEQ ID NOs210, l2, l4, 16, 18, 20, 22, 24, 26, or 36, provides a pro-stimulatory immune effect in a subject in need thereof as opposed to an anti-stimulatory or pro-regulatory effect. In some embodiments, use of an engineered or modified L-2 antibody comprising a light chain variable region having the sequence of one of SEQ ID NOs21 l, 13, 15, l7, 19, 21, 23, 25, 27, or 37, provides a pro-stimulatory immune effect in a subject in need thereof as d to an anti-stimulatory or pro-regulatory effect. In some embodiments, use of an engineered or ed anti-IL-2 antibody comprising a heavy chain variable region and a light chain variable region having the sequences of one of: SEQ ID NOs: 10 and 11; SEQ ID NOs212 and 13; SEQ ID NOs214 and 15; SEQ ID NOs216 and 17; SEQ ID NOs: 18 and 19; SEQ ID NOs22O and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27; or SEQ ID NOs: 36 and 37, es a pro-stimulatory immune effect in a subject in need thereof as d to an anti- stimulatory or pro-regulatory effect. In some embodiments, said use comprises the anti-IL-2 antibody.

In some embodiments, said use comprises the anti-IL-2 dy and an IL-2. In some embodiments, use comprises a complex of an anti-IL-2 antibody With an IL-2.

In some embodiments, use of an anti-IL—2 antibody sing a heavy chain variable region comprising heavy chain CDRl, CDR2 and CDR3 as set forth in amino acid sequences SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 tively, ; SEQ ID NOs250-52 respectively; SEQ ID -58 respectively; or SEQ ID NOs262-64 respectively, provides a pro-stimulatory immune effect in a subject in need thereof. In some embodiments, use of an anti-IL-2 antibody comprising a light chain sing light chain CDRl, CDR2 and CDR3 as set forth in amino acid sequences SEQ ID NOs24l-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID NOs265-67 respectively, provides a pro-stimulatory immune effect in a subject in need f. In some ments, use of an anti-IL-2 antibody comprising a heavy chain variable region comprising heavy chain CDRl, CDR2 and CDR3 as set forth in amino acid sequences SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively, ; SEQ ID NOs250- 52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID NOs262-64 respectively, and a light chain sing light chain CDRl, CDR2 and CDR3 as set forth in amino acid sequences SEQ ID NOs24l-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID NOs265-67 respectively, provides a pro-stimulatory immune effect in a subject in need f. In some embodiments, said use comprises the anti-IL-2 antibody.

In some embodiments, said use comprises the anti-IL-2 antibody and an IL-2. In some ments, use ses a complex of an anti-IL-2 antibody With an IL-2.

In some embodiments, use of an anti-IL—2 dy comprising a heavy chain variable region comprising heavy chain CDRl, CDR2 and CDR3 as set forth in amino acid sequences SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively, ; SEQ ID NOs250-52 tively; SEQ ID NOs256-58 respectively; or SEQ ID -64 respectively, provides a pro-stimulatory immune effect in a subject in need thereof as opposed to an anti-stimulatory or pro-regulatory effect. In some embodiments, use of an anti-IL-2 antibody comprising a light chain comprising light chain CDRl, CDR2 and CDR3 as set forth in amino acid ces SEQ ID NOs24l-43 tively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID NOs265-67 respectively, provides a pro-stimulatory immune effect in a subject in need f as opposed to an anti-stimulatory or gulatory effect. In some embodiments, use of an anti-IL-2 antibody comprising a heavy chain variable region comprising heavy chain CDRl, CDR2 and CDR3 as set forth in amino acid sequences SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively, ; SEQ ID NOs250-52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID NOs262- 64 respectively, and a light chain comprising light chain CDRl, CDR2 and CDR3 as set forth in amino acid sequences SEQ ID NOs24l-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID NOs265-67 respectively, provides a pro-stimulatory immune effect in a subject in need thereof, as opposed to an anti- stimulatory or pro-regulatory effect. In some embodiments, said use comprises the anti-IL-2 antibody. In some embodiments, said use comprises the anti-IL-2 antibody and an IL-2. In some embodiments, use ses a complex of an anti-IL-2 dy With an IL-2.

Thus, the engineered anti-IL-2 antibodies disclosed herein would be useful in adjusting immune cell populations and ng differential expansion of certain immune or cells in a method of treating a disease such as viral infection, bacterial infection, or cancer, or treating a condition such as IL-2 induced pulmonary edema, or ILinduced vascular leakage.

In some embodiments, sed herein is a method of immunizing of a subject, wherein said immunization comprises administration of a vaccine comprising an adjuvant, said adjuvant comprising an IL-2 antibody adjuvant. In some embodiments, an IL-2 antibody adjuvant comprises the anti-IL-2 antibody and IL-2, or comprises an L-2 dy complexed With IL-2. In some embodiments, an IL-2 antibody adjuvant comprises the anti-IL-2 antibody and IL-2. In some embodiments, an IL-2 antibody nt ses an anti-IL-2 antibody complexed With IL-2. In some embodiments, an IL-2 antibody adjuvant comprises an anti-IL-2 antibody.

In some embodiments, the subject being immunized is a mammalian subject. In some embodiments, the subject being immunized is a human. In some embodiments, the subject being zed has a ed immune .

In some embodiments of a method of immunization, the anti-IL-2 antibody comprise a heavy chain variable region having the sequence of one of SEQ ID NOs: 10, l2, l4, 16, 18, 20, 22, 24, 26, or 36. In some embodiments of a method of immunization, the anti-IL-2 antibody comprise a light chain variable region having the sequence of one of SEQ ID NOs: ll, 13, 15, l7, 19, 21, 23, 25, 27, or 37. In some embodiments of a method of immunization, the anti-IL-2 antibody comprise an anti- IL-2 antibody comprising a heavy chain variable region and a light chain variable region having the sequences of one of: SEQ ID NOs: 10 and 11; SEQ ID NOs212 and 13; SEQ ID NOs214 and 15; SEQ ID NOs:l6 and 17; SEQ ID NOs218 and 19; SEQ ID NOs220 and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27; or SEQ ID NOs236 and 37.

In some embodiments of a method of immunization, the anti-IL-2 antibody comprises an anti-IL-2 antibody comprising a heavy chain le region comprising complementarity determining region (CDR) l, CDR2 and CDR3, said CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively; SEQ ID NOs250-52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID -64 respectively. In some embodiments of a method of immunization, the anti-IL-2 antibody comprises and anti-IL-2 antibody comprising a light chain variable region comprising complementarity ining region (CDR) l, CDR2 and CDR3, said CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID - 43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259— 61 respectively; or SEQ ID NOs265-67 tively. In some embodiments of a method of immunization, the anti-IL-2 antibody comprises an L-2 antibody comprise a heavy chain variable region and a light chain variable region, each of said heavy chain variable region and light chain variable region comprises complementarity determining region (CDR) l, CDR2 and CDR3, wherein said heavy chain CDRl, CDR2 and CDR3 comprise amino acid ces of SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively; SEQ ID -52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID NOs262-64 respectively, wherein said light chain CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs24l-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID NOs265- 67 respectively.

In some embodiments of a method of immunizing a t, an immunization comprises administration of a vaccine comprising an adjuvant, said adjuvant comprising an IL-2 antibody adjuvant, said anti-IL-2 antibody comprising an anti-IL-2 antibody as disclosed herein. In certain embodiments, an IL-2 antibody nt comprises the anti-IL-2 antibody and IL-2, or comprises an anti-IL-2 dy complexed with IL-2. In some embodiments, of a method nizing a subject, a subject has a weakened immune system.

In some embodiments, a subject for immunization with a vaccine comprising an IL-2 antibody adjuvant comprises a subject suffering from a ion comprising a genetic position that increases likelihood of cancer in said t. In some embodiments, the genetic predisposition comprises a change in expression or activity of a gene product. In some embodiments, a genetic predisposition that increases the hood of cancer comprises a mutation in a tumor suppressor gene or a mismatch repair (MMR) gene, or a combination thereof. Many hereditary cancers are known in the art non-limiting examples include but are not limited to Hereditary Breast and Ovarian Cancer (HBOC) syndrome, Lynch syndrome (hereditary non-polyposis colorectal cancer), and Li-Fraumeni syndrome.

In some embodiments, a subject treated by a method disclosed herein for treating a disease or condition is further treated with one or more immune checkpoint inhibitors targeting one or more immune oints. In some embodiments, a subject is treated with said immune checkpoint inhibitors concurrently, before, or after treatment with said anti-IL-2 antibody. In some ments of a method of ng disclosed herein, an immune checkpoint comprises PD-l, PDL-l, CTLA-4, TIGIT, TIM—3, B7—H3, CD73, LAG3, CD27, CD70, 4—1BB, GITR, 0X40, SIRP—alpha , CD39, ILDR2, VISTA, BTLA, or VTCN—l, or a combination f.

As discussed above, in some embodiments, a therapeutic method of treatment as disclosed herein, further comprises an additional active agent comprising a checkpoint inhibitor. One d in the art would iate that a combination therapy comprising an anti-IL-2 antibody therapy in the presence or absence of IL-2, and additionally comprising a checkpoint tor may utilize any of the therapeutic itions or formulations comprising an anti-IL-2 antibody +/- IL-2, and a checkpoint inhibitor as provided herein. In some embodiments, at least two checkpoint inhibitors are used in a combination therapy. id="p-204" id="p-204" id="p-204"

[0204] Embodiments of this application include: An isolated anti-IL-2 antibody, wherein the antibody comprises a heavy chain variable region comprising the sequence of one of SEQ ID NOs: 10, l2, l4, 16, 18, 20, 22, 24, 26, or 36.

An antibody including an dy comprising an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, a F(ab’)2, a minibody, a diabody, a triabody, a nanobody, or a single domain dy. id="p-207" id="p-207" id="p-207"

[0207] An IgG comprising (a) an IgGl, IgG2, IgG3, or an IgG4; (b) a heavy chain comprising a mutation that that reduces binding to a ch receptor (chRs); (c), a lambda or kappa light chain; or (d) any combination of (a)-(c) thereof.

A ition comprising the isolated anti-IL-2 antibody and a ceutically acceptable carrier.

An isolated anti-IL-2 antibody, wherein the antibody comprises a light chain variable region comprising the sequence of one of SEQ ID NOS: 1 l, 13, 15, l7, 19, 21, 23, 25, 27, or 37. id="p-210" id="p-210" id="p-210"

[0210] An isolated anti-IL-2 antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region having the sequences of one of: SEQ ID NOs210 and 11; SEQ ID NOs212 and 13; SEQ ID NOs:l4 and 15; SEQ ID NOs216 and 17; SEQ ID NOs218 and 19; SEQ ID NOs220 and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27; or SEQ ID NOs236 and 37. [021 1] An ed anti-IL-2 antibody, wherein the antibody comprises a heavy chain variable region having complementarity determining region 1 (CDRl), CDR2 and CDR3, said CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively; SEQ ID NOs250-52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID NOs262- 64 respectively.

An isolated anti-IL-2 antibody, wherein the antibody comprises a light chain variable region having complementarity determining region 1 , CDR2 and CDR3, said CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs24l-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID -6l respectively; or SEQ ID NOs265- 67 respectively.

An isolated anti-IL-2 antibody, wherein the antibody comprises a heavy chain variable region comprising mentarity determining region 1 (CDRl), CDR2 and CDR3, said CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs238-40 tively; SEQ ID -46 respectively; SEQ ID NOs250-52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID NOs262- 64 respectively; and a light chain variable region having complementarity ining region 1 (CDRl), CDR2 and CDR3, said CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs24l-43 respectively; SEQ ID -49 respectively; SEQ ID NOs253-55 tively; SEQ ID -6l respectively; or SEQ ID -67 respectively.

An isolated polynucleotide sequence encoding a heavy chain le region of an anti-IL-2 antibody, wherein the heavy chain variable region comprises the amino acid sequence of one of SEQ ID NOs210, 12, 14, 16, 18, 20, 22, 24, 26, or 36. id="p-215" id="p-215" id="p-215"

[0215] A vector comprising a polynucleotide sequence described . A host cell comprising a vector described herein.

An isolated polynucleotide sequence encoding a light chain variable region of an anti-IL-2 antibody, wherein the light chain variable region comprises the amino acid sequence of one of SEQ ID NOs211, 13, 15, 17, 19, 21, 23, 25, 27, or 37. id="p-217" id="p-217" id="p-217"

[0217] An isolated polynucleotide sequence encoding a heavy chain variable region of an anti-IL-2 antibody, wherein the heavy chain variable region comprises the amino acid sequence of one of SEQ ID NOs: 10, l2, l4, 16, 18, 20, 22, 24, 26, or 36, and encoding a light chain variable region of an anti- IL-2 antibody, wherein the light chain variable region comprises the amino acid sequence of one of SEQ ID NOs211, 13, 15, 17, 19, 21, 23, 25, 27, or 37. id="p-218" id="p-218" id="p-218"

[0218] An isolated polynucleotide sequence encoding a scFv, said polynucleotide sequence comprises the sequence of one of SEQ ID NOS: 1, 2, 3, 4, 5, 31, 32, 33, 34, or 35.

A method of producing a heavy chain variable region of an anti-IL-2 antibody, said method comprises the step of culturing a host cell comprising a vector disclosed herein, under conditions conducive to expressing said vector in said host cell, thereby ing the heavy chain variable region of the anti-IL-2 antibody. id="p-220" id="p-220" id="p-220"

[0220] A method of producing a light chain variable region of an anti-IL-2 antibody, said method comprises the step of culturing a host cell under conditions conducive to expressing said vector in said host cell, thereby producing the light chain variable region of the anti-IL-2 antibody.

A method of producing an anti-IL-2 antibody sing a heavy chain variable region and a light chain variable region of an anti-IL-2 dy, said method comprises the step of culturing a host cell under conditions conducive to expressing said vector in said host cell, thereby producing the heavy chain variable region and the light chain variable region of the anti-IL-2 antibody.

A method of promoting differential growth of immune cells in a subject, comprising the step of stering a composition comprising an anti-IL-2 antibody, thereby ing differential growth of immune cells in the subject. In some embodiments, the composition ses the anti-IL- 2 antibody and IL-2, or the anti-IL-2 dy complexed with IL-2.

A method of treating a t with cancer through induction of ential growth of immune cells, comprising the step of administering a ition comprising an anti-IL-2 antibody, thereby treating a subject with cancer.

A method of treating a disease or a condition in a subject, comprising the step of administering to the subject a composition comprising an anti-IL-2 antibody wherein said antibody facilitates expansion of subsets of immune cells and decreases undesirable effects caused by IL-2, thereby treating said e or condition in said subject.

In some embodiments, the disease comprises a viral infection, a bacterial infection, or a cancer. In some ments, the viral ion is caused by SARS CoV-2,; rus; rotavirus; hepatitis virus A, B, C, D, or E; rabies virus; West Nile virus; enterovirus; echovirus; coxsackievirus; herpes simplex virus (HSV); HSV-2; lla-zoster virus; mosquito-borne viruses; arbovirus; St.

Louis encephalitis virus; California encephalitis virus; lymphocytic choriomeningitis virus; human immunodeficiency virus (HIV); poliovirus; zika virus; rubella virus; cytomegalovirus; human papillomavirus (HPV); enterovirus D68; severe acute respiratory syndrome (SARS) coronavirus; Middle East respiratory syndrome coronavirus; Epstein-Barr virus; influenza virus; respiratory syncytial virus; polyoma viruses including JC virus; BK virus); Ebola virus; Dengue virus; or any 19236139_1 (GHMatters) P119595.NZ combination thereof. In some embodiments, the condition comprises a weak immune system and said treatment prophylactically boosts the immune system.

In some embodiments, the condition comprises IL-2 induced pulmonary edema.

In some embodiments of a method disclosed herein, the immune cells comprise one or more of naive T cells, memory T cells, CD8+ T cells, NK cells, or l Killer T cells.

In some embodiments, the rable effect caused by IL-2 ses one or more of activation of tory T cells, apoptosis of CD25+ T effector cells, IL-2 induced pulmonary edema, pneumonia, or ILinduced vascular leakage.

In some ments, an anti-IL-2 antibody disclosed herein inhibit IL-2 binding to CD25. id="p-230" id="p-230" id="p-230"

[0230] A method of zing of a subject, wherein said immunization ses administration of a vaccine comprising an adjuvant, said adjuvant comprising an IL-2 antibody adjuvant.

In some embodiments, the IL-2 antibody nt comprises the anti-IL-2 antibody and IL- 2, or comprises an anti-IL-2 antibody complexed with IL-2.

In some embodiments, a subject is an animal or a human. In some embodiments, the subject has a weakened immune system.

In some embodiments of a method disclosed , the immune cells are CD8+ cells or NK cells.

In some embodiments of a method disclosed herein, said L-2 antibody comprise a heavy chain variable region having the sequence of one of SEQ ID NOs: 10, l2, l4, l6, 18, 20, 22, 24, 26, or 36.

In some embodiments of a method disclosed herein, the anti-IL-2 dy comprise a light chain variable region having the sequence of one of SEQ ID NOs: ll, 13, 15, l7, 19, 21, 23, 25, 27, or 37.

In some embodiments of a method disclosed herein, the anti-IL-2 antibody comprise a heavy chain variable region and a light chain variable region having the sequences of one of: SEQ ID NOs210 and 11; SEQ ID NOs212 and 13; SEQ ID NOs214 and 15; SEQ ID NOs216 and 17; SEQ ID NOs218 and 19; SEQ ID NOs220 and 21; SEQ ID NOs222 and 23; SEQ ID NOs224 and 25; SEQ ID NOs226 and 27; or SEQ ID NOs236 and 37.

In some embodiments of a method disclosed herein, the anti-IL-2 antibody comprise a heavy chain variable region comprising complementarity determining region (CDR) l, CDR2 and CDR3, said CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively; SEQ ID NOs250-52 respectively; SEQ ID NOs256-58 respectively; or SEQ ID NOs262-64 respectively.

In some embodiments of a method disclosed herein, the anti-IL-2 antibody comprise a light chain variable region comprising complementarity ining region (CDR) l, CDR2 and CDR3, said CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs24l-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID -67 tively.

In some embodiments of a method disclosed herein, the anti-IL-2 antibody comprise a heavy chain variable region and a light chain variable region, each of said heavy chain variable region and light chain variable region comprises complementarity determining region (CDR) l, CDR2 and CDR3, wherein said heavy chain CDRl, CDR2 and CDR3 comprise amino acid ces of SEQ ID NOs238-40 respectively; SEQ ID NOs244-46 respectively; SEQ ID -52 respectively; SEQ ID NOs256—58 respectively; or SEQ ID NOs262-64 tively, wherein said light chain CDRl, CDR2 and CDR3 comprise amino acid sequences of SEQ ID NOs24l-43 respectively; SEQ ID NOs247-49 respectively; SEQ ID NOs253-55 respectively; SEQ ID NOs259-6l respectively; or SEQ ID NOs265-67 respectively.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a ity of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an in?exible limitation on the scope of the invention.

Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically sed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as dual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. id="p-242" id="p-242" id="p-242"

[0242] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the ted range. The s "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

A skilled artisan would iate that the term "about", may encompass a deviance of between 0.0001-5% from the indicated number or range of numbers. In some instances, the term "about", may ass a deviance of between 1 -10% from the indicated number or range of numbers. In some instances, the term "about", encompasses a deviance of up to 25% from the indicated number or range of numbers.

EXAMPLES EXAMPLE 1 This example describes generation of modified anti-IL—2 antibodies based on embodiments of the antibodies generated. The exemplification of generating ed anti-IL-2 antibodies is based on a sub-set of the antibodies disclosed herein. The description and results presented in Example 1 are exemplary and do not limit the generation of modified anti-IL-2 antibodies disclosed throughout this application.

Library Design A library was designed to introduce variation into the sequence of JES6.l. Amino acid sequences for the heavy chain variable region and light chain variable region of JES6.l are shown in SEQ ID N026 and SEQ ID N027 respectively. Brie?y, three ons were varied with a codon encoding all amino acids (codon NNS). The design of the library allowed for one mutation in both CDRs L3 and H3, as well as a one mutation in one of the ing CDRs: H1, H2, or L2. CDRs were defined by meeting either the IMGT or ABR (Kunik et al., 2012) definitions. CDR residues that are ved (based on Blast search against the PDB database) or don’t form specific interactions with mouse IL-2 (mIL-2) in the crystal structure of the mIL2-JES6.1 complex (PDB 4YQX), were ed from ion. The theoretical size of the library was of 1.38E+7 variants.

Library Selection Screening and Selection Using Yeast Smface Display displayed scFv libraries were grown in a SDCAA selective medium and induced for expression with 2% w/v galactose at 30 OC overnight according to established protocols. The library was incubated with 100nM of recombinant human IL—2 with a 6xhis tag (hIL—2-His) (Reprokine, Israel) in PBS 0.1% BSA for 1 hour, then washed three times with PBS 0.1% BSA and labeled with ?uorescent d antibodies mouse anti TC (Santa Cruze, USA) and mouse monoclonal anti-His APC (Miltenyi Biotec, Germany. cat 0020130782). Post labeling the y was sorted on BioRad S3e Fluorescence Activated Cell Sorter for high affinity binders to recombinant human IL-2. Isolated clones from the ?nal sort were sequenced by tion of plasmid DNA from the yeast clones using a Zymoprep kit (Zymo Research, USA) and the DNA was sequenced.

Ko?"Selection To select for binders with improved off rate the clones of round 2 of selection were ted for 15min with 10nM 6xhis tag ), then the yeast were washed 3x with lml PBS 0.1% BSA and incubated for 5 minutes, 4h, 6h, and 24h with 100nM unlabeled IL-2. At the indicated time points the yeast were washed and labeled with Myc-FITC (Santa Cruze, USA) and monoclonal anti-His APC (Miltenyi Biotec, Germany. cat 0020130782), and sorted on a Se3 as described above.

IgG tion JES6. lw.t. was bought from Thermo Fisher (cat: 2-81).JES6.1.RMC was cloned as a rat Fv with a mouse IgG2a constant region, and produced by GeneScript antibody production services ript NJ, USA). BDGl7.0014 was cloned into human IgG1 constant region and produced by GeneScript antibody production services. Amino acid sequences for the heavy chain variable region and light chain variable region of JES6.1.RMC are shown in SEQ ID N028 and SEQ ID N029 respectively. All other antibodies were generated as described below. id="p-254" id="p-254" id="p-254"

[0254] Reformatting [025 5] Selected scFv clone was reformatted to human IgG1 . The sequences of the light chain (LC) and heavy chain (HC) variable regions were optimized to mammalian codon usage and ordered as genblocks (GB) from IDT (Integrated DNA Technologies. Coralville, Iowa USA). The GB were cloned using standard cloning techniques into pSF-CMV-HngGl_HC (HC plasmid) and pSF-CMV- HuLambda_LC (LC plasmid) (Oxford genetics, Oxford UK). When indicated, the variable Heavy chain was cloned into pSF-CMV-HngGl_HC_LALA (HC plasmid) in which the DNA coding for L234 and L235 of the heavy chain was mutated to alanine codons (L234A, L235A) IgG Expression Expi-CHO cells (Thermo Fisher Scientific, USA) were transfected with LC and HC plasmids at a ratio of 2:1 and expression was done according to the cturer‘s instructions. Brie?y: 50ml Expi-CHO cells were cultured at 37°C, l20rpm, 8% C02 to a density of 6x106 cells/ml. Then, 50pg of heavy chain and light chain expression plasmids at a ratio of 122 were transfected into the CHO cells. Post transfections, a booster enhancer and feed were added to the culture, and growth conditions were d to 32°C, 120rpm, 5% C02. The cells were ted 10 days after transfection. The IgGs were purified from the supernatant using proteinA beads (Tosoh Bioscience GmbH, Germany), followed by size exclusion chromatography (SEC) purification on ex 200 10/300 increase column, with PBS as mobile phase (GE healthcare, USA).

Sequences DNA sequences encoding scFv of clone 1 (17.021) is shown in SEQ ID N021. DNA sequences ng scFv of clone 2 (17.022) is shown in SEQ ID N022. DNA sequences encoding scFv of clone 4 3) is shown in SEQ ID N023. DNA sequences encoding scFv of clone 5 (17.030) is shown in SEQ ID N024. DNA sequences encoding scFv of clone 6 (17.035) is shown in SEQ ID N025.

Amino acid sequences for the heavy chain variable region and light chain variable region of original JES6_1 starting sequence and the various L-2 clones are shown in the table below and in FIGS. 11 and 12.

Table 2: VH and VL Amino Acid Sequences of a Sub-Set of Anti-IL-2 Clones.

Reion SEQ ID N0212 SEQ ID N0213 17.023 SEQ ID N0214 SEQ ID N0215 17.030 SEQ ID N0216 SEQ ID N0217 17.035 SEQ ID N0218 SEQ ID N0219 Measurements of IgG Binding to Human IL-2 The SPR analysis was done on Biacore 200 (GE healthcare, USA) on CM5 chips (cat2br10005-30, GE healthcare, USA). The chip was crosslinked with primary capture Ab against human IgG (Cat: br39, GE healthcare, USA) or primary e antibody against mouse IgG (Cat: BR38, GE healthcare, USA) to a target of 8000RU. After linking of the primary Ab, the mouse and human tested antibodies were immobilized on the primary Ab to a target of imately additional 500RU. JES6.1 was cross-linked directly to the CM5 chip. Human IL-2 (Cat: 60568, Reprokine, Israel) analyte was streamed in HEB-EP or PBS 0.05% tween-20 (PBS-T) buffer at concentrations ranging from 128nM to 0.03nM in a series of two-fold or three-fold dilutions, one tration for each cycle. Mouse IL-2 (Cat: RKP04351, Reprokine, ) was streamed in HEB-EP or PBS-T buffer at trations g from 0.5nM to 40nM. At the end of each cycle the analyte and tested antibody were stripped from the chip using 3M MgClz and new tested Ab was loaded on the chip as described above. When indicated, instead of stripping the antibodies, kinetics was determined by injecting series of analyte concentrations in one cycle by the Single-cycle kinetics method. Binding cs were determined by the 121 g model using the Biacore T200 evaluation software.

Binding of IgGs to cynomolgus monkey IL-2 The SPR analysis was done on Biacore 200 (GE healthcare, USA) on CM5 chips (cat:br10005-30, GE healthcare, USA). The chip was inked with primary capture Ab t human IgG (Cat: br39, GE healthcare, USA) to a target of 5000RU and cynomolgus monkey IL-2 (cIL-2) was tested by the multi-cycle method in the same conditions described above.

SEC analysis.

To analyze the IgGs, 100ug samples were loaded on a Superdex 200 10/300 increase column (GE healthcare, USA) at a flow rate of 0.8ml/min on a GE AKTA Explorer chromatography system (GE healthcare, USA). ring of antibody retention time was done at 280nm.

Testingfor Specific Binding to CD25 and CD122 To test specific binding to CD25, BDGl7.023 was immobilized to the CM5 chip to a target RU of approximately 300RU as described above. Subsequently, 50nM IL-2 was injected till the BDGl7.023 or control antibody were saturated. Then the Ab-IL-2 complex was washed with PBS- T buffer for 10 sec and 1000nM of CD25 was injected and monitored for response.

To test speci?c binding to CD122, BDGl7.023 was immobilized to the CM5 chip to a target RU of approximately 300RU-500RU as described above. Subsequently, 50nM hIL-2 was injected till the 023 antibody was saturated with hIL-2. Next the Ab-IL-2 complex was washed with PBS-T buffer for 10 sec and 1000nM of CD122 was injected and response was monitored.

To test specific binding of the humanized antibodies IL-2 complex to CD122 and CD25, antibodies BDG17.038, BDG17.043, BDG17.053, BDG17.054, BDG17.067, BDG17.069 (See, Tables 6 and 7 of Example 2 for sequence information for these clones) were immobilized to an a capture antibody attached to CM5 chip channel to a target RU of imately 300RU as described above. Subsequently, 50nM IL-2 was ed till the respective antibody was saturated with hIL-2.

WO 61287 2021/051267 Then the Ab-IL-2 complex was washed with PBS-T buffer for 60 sec and 1000nM of CD25 was injected and monitored for se. Subsequently, running buffer was injected for 60 seconds to reach a steady baseline, and then 1000 nM of CD122 was injected for 30 seconds in a ?ow rate of 30 ul/min. To test CD122 binding, the same experiment was repeated in reverse order, with CD122 injected firs ?owed by ion of CD25.

DSF analysis of IgG Tm To determine the T-onset and Tm of the humanized anti-hIL-2 antibodies, antibodies were diluted to 0.5 mg/ml in PBS and analyzed using NanoDSF Prometheus NT.48 (Nanotemper, Germany) in a temperature elevation rate of 1°C/min. id="p-274" id="p-274" id="p-274"

[0274] In Vivo ments Treatment ofMice with the IL-2/Ab Complex Groups of six male C57BL/6 mice, 7-8 weeks old, were injected eritoneally (i.p.) with BDGl7.023/hIL-2 or JES6. l/mIL-2 immune complex daily, for four consecutive days. PBS and free hIL-2 or mIL-2 served as controls. At the end of the fourth day the mice were sacrificed, s were harvested and homogenized to a single cell suspension. The cells were filtered, centrifuged (400g for minutes) resuspended in 5ml PBS to a final concentration of 5x106lymphocytes/ml. The experiment was done in accordance with the guidelines of the national council for Institutional Animal Care and Use Committee ) in Israel.

Groups of six male C57BL/6 mice, 7-8 weeks old, were injected intraperitoneally (LB) with BDGl7.038/hIL—2, BDGl7.043/hIL—2, BDGl7.054/hIL—2, BDGl7.038/hIL—2 or e control/hIL-2 immune complex daily, for four consecutive days. To form the complex, 10ug of the antibody was pre-incubated with 0.5ug of hIL-2 for 30 s at 37°C before the injection. At the end of the fourth day the mice were sacrificed, spleens were harvested and homogenized to a single cell suspension. The cells were filtered, centrifuged (400g for 5 minutes) resuspended in 5ml PBS to a final concentration of 5x106 lymphocytes/n11. The experiment was done in accordance with the guidelines of the national council for Institutional Animal Care and Use Committee (IACUC) in Israel.

BI6F10 marine melanoma tumor xenograft model Female C57BL/6 mice were inoculated subcutaneously in the right rear ?ank region with (2 x 105) B 16-F10 tumor cells. Five days post inoculation when the tumor volume reached ~30-50mm3, the mice were randomized into experimental groups (n=10 per group) and injected intraperitoneally daily with single doses of lOug anti-IL-2 antibody/ 1 ug hIL-2 complex of the indicated antibodies or with PBS l for four consecutive days. The mice were monitored for tumor volume growth, body weight loss and for non-specific clinical signs throughout the experiments.

Determination ofImmune Cell Population By FACS In order to identify immune cell populations, spleen lymphocytes were d with the antibodies described below according to the manufacturer‘s instructions. Regulatory T cells ) were designated as cells labeled as CD45+/CD3+/ CD4+/ CD25+/ FoxP3+. Memory phenotype effector T cells (MP CD8+): CD45+/CD3+/ CD8+/ CD44+/ IL—2RB(CD122)+. Natural killer cells (NK): CD45+/CD3'/ CD49b+/ NKl. 1(CD161). Natural killer T cells (NKT): CD45+/CD3+/ CD49b+/ NKl.l(CDl6l). Positive cells frequency and number were calculated from the raw data acquired on the flow cytometer.

Table 3: Marker/Labeled dies t Catalog No. Channel Excitation on Concertation Dilution i " ‘ Thermo Filter Filter (mg/ml) (11g) Fisher : 3 [test Scientific EAntl-MO/RT 11577} EFOXPs FJK-16S FLl 488 82 5525/50 0'5 1 gFITC100UG 3 3 3 3 3 CD25 Monoclonal iAntibody (CDZS- 12‘0251‘ FL2 488 $575/30 0.2 0.125 4E3), PE 100T 82 2 2 ECD49b (Integrin alpha 2) iMonoclonal 61—5971— FL3 488 0.2 0.5 Antibody (DXS), 82 E620/30 610/100ug ECD4 anti mouse, EPerCP/CyanineSS BLG FL4 488 $1001ug 100540 i695/30 0.2 i025 CD122 (IL-2RB) ian? mouse, PE' BLG FL5 488 i755LP 0.2 0.25 gCyanine7/100 pg 3 123216 ‘ NK1.1 Monoclonal 3Antib°dY(PK136)’ 317'5941' FL6 638 2660/20 0.2 30125 gAPC/100ug 82 2 2 2 3CD3 Monoclonal Antibody (17A2), 3Alexa Fluor 700, FL7 638 725/20 0.2 0.25 eBioscienceTM / 3 3 3 3 CD44 Monoclonal 3Antibody (1M7) 247_ APGeFluor 780} (8)341 _ FL8 638 2755LP 0.2 20.25 100ug CD8a Monoclonal Antibody (SS-6.7), 2511!)" Bright 436, 62'0081' FL9 405 450/50 0.2 0.25 eBioscienceTM/ 100 82 CD45 Monoclonal Antibody (30- 69 0451_ _ 2F11), eFluor 506, FL10 405 525/50 0.2 0.5 3 . . 2 82 2 3 3 eBlosclenceTM/ 2 2 2 2 : 2 2 3 100ug : : 2222222222 2222222222222222222222222222222222 222222222222222222222222222 2 22222222222222222222222222222:222222222222222222222222222 2 222222222222222222222222222222222222 RESULTS JES6.] binds mouse strongly but does not bind the human IL-2 JES6.1 has been reported to bind mouse IL-2 (mIL-2) at a KD of 5.6nM. To test if JES6.1 could bind human IL-2 (hIL-2), JES6.1 antibody was tested by SPR on eT200. JES6.1 was cross-linked directly to the CM5 chip, then human IL-2 or mouse IL-2 es were streamed at concentrations ranging from 0.5nM to 128nM or 0.5 to 16nM tively. As can be seen in , when tested with human IL-2, the JES6.1 showed no apparent change in response unit (RU). On the other hand, when mouse IL-2 served as analyte, a robust se was apparent (), indicating that JES6.1 binds mouse IL-2 strongly but does not bind human IL-2. The ment was repeated with the JES6.1RMC antibody chimera which was sed as a JES6.1 rat FV with a mouse constant region as described herein. The JES6.1RMC was immobilized on the CM5 chip using the GE antibody capture kit. Streaming hIL-2 at a concentration ofup to 100nM resulted in no change in RU, indicating no binding to the human IL-2 (). To test r the JES6.1RMC chimera retained its mIL-2 binding properties like the JES6.1 above, it was tested for binding to mouse IL-2.

Streaming of mIL-2 at a concentration of 0.5nM to 320nM resulted in large change in RU, ting robust binding (). These results indicate that JES6.l and JES6.1RMC bind mouse IL-2 strongly but show no apparent g to human IL-2. Analysis of the JES6.1RMC binding kinetics to both hIL-2 and mIL-2 are shown below.

Table 4: Binding Kinetics of lIES6.1RMC.

Mouse IL-2 Human IL-2 BDG 1.3*10A—10 4.8*10’\5 63*10-5 ND. ND JES6.1RMC (17.006) Changing Binding Speci?cityfrom Mouse IL-2 to Human IL-2 To change binding specificity from mouse IL-2 to human IL-2, JES6.l was cloned as a scFv into a yeast display vector. The scFv format of JES6.l expressed well on the yeast surface as indicated from the y terminal myc tag labeling (FIGS. SA-SC). Incubating lOOnM JES6.l in IgG format with YSD clones expressing mouse IL-2 resulted in strong binding (FIGS. SA-SC). However, in correlation with the SPR results shown above, incubation of JES6.l YSD clones with up to luM labeled human IL-2 showed no se in ?uorescence, indicating that the JES6.l scFv does not bind hIL—2. id="p-289" id="p-289" id="p-289"

[0289] Based on the JES6.l scFv, a nesis library was generated as described above. Brie?y, the YSD library was selected against recombinant human IL-2 as described above. The mutant library went through one round of MACS selection against luM of human IL-2 and additional round of FACS selection against luM of human IL-2. The top 0.2% clones were selected. Subsequently, the library underwent two additional rounds of selection specifically aimed at ing the koff properties of the ed clones as described above. For 3Id round of selection the yeast were incubated with lOnM His-tagged hIL-2 for 15 minutes at room temperature, then the yeast were washed of the hIL-2 and incubated for 5 s with lOOnM unlabeled IL-2 at room temperature.

The 4th round was done in a similar fashion but post labeling and wash, the yeast were incubated in fold of initial volume in PBS for 24 hours. In the 5th round, the yeast were d and washed, and then incubated with lOOnM unlabeled IL-2 at room temperature for six hours. After ?ve rounds of selection the clones were isolated. Five YSD clones that gained binding to hIL-2 (FIGS. 6A-6B) were sequenced. In addition, these clones were tested for specificity by ng with a mixture of 500nM e TNFR2, and 500nM 0X40 and 500nM PD1. As can be seen in FIGS. 6A-6B, these clones are specific to hIL-2 and do not bind any of the other proteins.

Expression ofBDG] 7.023 Subsequent to YSD characterization, clone #4, which showed significant binding to hIL-2, was reformatted to human chimeric IgGl (BDG17.023) with rat FV and human Fc chimera. The rat variable domain was subcloned into two separate sion vectors, pSF-CMV-HngG1_HC and pSF-CMV-HuLambda_LC as described above. The IgG was expressed in O cells as described above. The purified IgGs were >95% pure as evident from a SDS PAGE is. Size exclusion chromatography of BDG17.023 on superdex200 10/300 showed two main peaks: the first peak with a retention time of ~9.2ml (0.36CV) was typical of large aggregate and the second peak with ion of approximately ~12.6ml (0.528CV) was typical of an ordinary human hIgGl. Peak integration of these SEC runs showed 11% and 89% respectively (.

Binding Kinetics ofBDG] 7. 023 To determine BDG17.023 binding kinetics and affinity to mIL-2 and hIL-2, the IgG was analyzed by SPR on a BIAcore T200 using the GE capture antibody kit as bed above. As shown in FIGS. 8A-8B, BDG17.023 binds hIL-2 with an affinity of approximately 8x10A-11, with a on rate of 1.3*10’\7 and off rate of 1*10A-3. BDG17.023 also showed binding to mIL-2 with a much lower affinity of approximately 2.5x10A-6.

Table 5: Kinetic arameters of BDG17.023.

Antibody ka(1/Ms) kd(1/s) ka(1/Ms) kd(1/s) Receptor Discrimination ofthe BDG17.023-hIL-2 Complex JES61-mIL-2 complex was reported to bind specifically to CD25 but not CD 122. It was shown that the JES6. 1 -mIL-2 complex bound to a SPR chip could bind CD25 but not CD122. To test whether BDG 17.023-hIL-2 complex can discriminate n binding to CD25 and CD122, a similar experiment was performed as described above. As shown in a control antibody complexed with hIL-2 binds human CD25 but could not bind CD 122. In contrast, the BDG17.023-hIL-2 complex was found to bind CD122 but not CD25. This result indicates that although BDG17.023 is derived from JES6.l, the JES6. l-mIL-2 complex and the BDGl7.023-hIL-2 complex show a very different IL-2 receptor preference, possibly through binding of different epitope of the mIL-2 and the hIL-2 respectively. Alternatively, different allosteric effects may be induced on the mIL-2 and hIL-2 that affects binding preference to the IL-2 ors.

In vivo Characterization 0fBDG17. 023 In vivo administration of JES6.l complexed with mIL-2 resulted in robust proliferation of regulatory T cells and much smaller eration of effector T cells, y shifting the MP regs ratio s immune ssion. Since the BDGl7.023-hIL-2 x showed preference to binding CD122 and excluded CD25 from binding in an SPR biochemical assay, it is predicted that the 023 -hIL-2 complex would enhance proliferation of CD8+ effector cells and NK cells in vivo. Human IL-2 can cross react with the mouse IL-2 receptors, thus the BDGl7.023- hIL-2 complex was administered to C57BL/6 mice to test its effect in vivo as described above. Brie?y, the 17.023 antibody-hIL-2 complex was incubated with hIL-2 at a 1:1 molar ratio and injected intraperitoneally to C57BL/6 mice daily, for four consecutive days. As a control. JES6.l-mIL-2 complex, hIL-2 alone, or mIL-2 alone was also administered in a similar fashion. On the fifth day the mouse spleens were harvested, cells were labeled and analyzed by FACS as bed above.

As can be seen in FIGS. 10A-10D, BDGl7.023 shows pronounced effect in inducing proliferation of MP CD8+ cells and NK cells, while there was much smaller effect on CD4+ Tregs.

On the other hand, JES6.l showed a much different effect in accordance with its reported anti- matory effect. These results demonstrate that in agreement with the binding data and in contrast to JES6.l, BDGl7.023-IL-2 complex has a strong stimulatory effect on the immune system in vivo, as opposed to an anti-stimulatory or pro-regulatory effect.

EXAMPLE 2 This example presents results on further ion of human IL-2 binder and generation of humanized antibodies.

Additional selection strategy was used to select human IL-2 binder under alternative selection pressure. Brie?y: the library went through one round of MACS selection against luM of human IL- 2 and additional four rounds of FACS selection against 100nM of human IL-2. After the first round of FACS selection, all binders were selected, followed by selection of the top 0.5%, top 0.5% and top 0.1% of the binders. After five rounds of selection, clone C#7 (l73R5Cl-l7.002) (SEQ ID N0228) was isolated, verified for binding and sequenced.

Antibody Humanization Clone C#7 (173R5C1-17.002) was selected as template for humanization. A human template was chosen using the Schrodinger BioLuminate ‘Antibody Humanization: CDR ng’ CDR tool (Kai Zhu, Tyler Day et al., Antibody structure determination using a combination of homology ng, energy-based refinement, and loop prediction. Proteins: ure, Function and Bioinformatics, 82, 8, 8 2014), and the PDB entry of JES6-1 was used as a query (4YQX; Jamie B.

Spangler, Jakub Tomala et al., Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational isms. Immunity, 42, 5, 5 2015). PDB entry 5118 (Alexey Teplyakov, Galina Obmolova et al., Structural diversity in a human antibody germline library. mAbs, 8, 6, 8 2016) was chosen as it had the best score with regard to a combination of framework identity of the L and H chains and stem ry. Mutations were introduced to positions that either interact (within 5A radius) with the CDR regions (according to IMGT numbering scheme), or the antigen in 4YQX. The variability on these positions was selected to include amino acids of the human te (5118) as well as the mouse query (4YQX). In addition, due to icant structural changes between H1 of the query and the template, the option of complete transition between H1 of 4YQX and 5118 was introduced. This y had approximately 1300 ent ts.

The library underwent selection by FACS for two . In the first round, the library was labeled with 5nM hIL-2 and the top 5% binding clones were sorted. In the second round, the library was labeled with 1nM hIL-2 and the top 5% of the clones were sorted. The clones were sequenced after selection and clone C#8 (173.2A.C6-17.014) (SEQ ID NO:31) was used as a template for affinity maturation.

Humanized Antibodies Af?nity Maturation CDR positions (IMGT/ABR definition) that were ted to have a high rate of somatic hypermutation were selected for variation. Additional positions in L3 that are presumed to interact with the antigen based on JES6-1, as well as all of H3, were also targeted for variation. The variation was based on ce conservation of all positions except for H3 which was the DHY codon. The theoretical diversity of this library was 3.21x1012.

Based on the clone C#8 (173.2A.C6-17.014) another library was constructed, in which all CDR ons were explored using the NNS degenerate codon that encodes all amino acids. Variants of 2-3 mutations, up to one on each CDR were screened. The theoretical size of such a library is 2x106 double mutants and 1x109 triple mutants.

The humanization affinity maturation libraries generated above were pooled together for selection. Brie?y, in the first round the pooled YSD libraries were labeled with 10nM hIL-2 and selected on MACS. In the second round the yeast were labeled with 0.1nM hIL-2 and selected using MACS. In the third round of selection, the yeast were labeled with 0.1nM hIL-2 and all binders were selected on FACS. For the fourth and fifth round of selections, the yeast were labeled with 10nM hIL- 2 and competed with 100nM unlabeled hIL-2 for 24 hours and 48 hours respectively. Subsequently, the yeast were sorted on FACS to select all the binders. The final round of selection was done in a similar n to the fourth and fifth rounds, but post labeling and wash, the yeast were ted in 1000 fold of l volume in PBS at room ature for a week. id="p-309" id="p-309" id="p-309"

[0309] After selection, the clones were isolated, veri?ed for g and sequenced. Amino acid sequences for several clones are shown below. Alignment of Heavy Chain and Light Chain variable regions, and CDR regions of a subset of clones is presented in Figs. 13A and13B.

Table 6: VH and VL Amino Acid Sequences of a Sub-Set of Anti-IL—2 Clones.

Clone Heavy Chain Variable Region Light Chain Variable Region 17.038 SEQ ID NO:20 SEQ ID NO:21 —17.043 SEQ ID NO:22 SEQ ID NO:23 17.014 —17.067 —17.069 id="p-311" id="p-311" id="p-311"

[0311] It should be noted that clones 17.066, 17.067, and 17.069 comprise the LALA mutation (L234A, L235A mutations).

CDR sequences for certain selected clones are shown below.

Table 7: CDR Amino Acid Sequences of Anti-IL—2 Clones.

Heavy Chain Light Chain -CDRl CDR2 CDR3 CDRl CDR2 CDR3 17.014 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID N0238 N0239 N0240 N0241 No:42 No:43 17.038 SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID No:44 N0245 N0246 N0247 N0248 N0249 17.043 —SEQ ID __———_— N056 N057 N058 N059 N0260 N0261 N0262 N0263 N0264 N0265 N0266 N0267 N0244 N0245 NO 46 N0247 N0248 NO 49 N0250 N0251 NO 52 N0253 N0254 NO 55 N0262 N0263 N0264 N0265 N0266 N0267 Table 8: ength Amino Acid Sequences of a Subset of Anti-IL-2 Clones.

BGD-Clone Heav Chain (LALA) Li_ht Chain BDG17.066 SEQ ID NO 68 SEQ ID NO: 69 BDG17.067 SEQ ID NO: 70 SEQ ID NO 71 BDG17.069 SEQ ID NO: 72 SEQ ID NO. 73 Binding Kinetics ofhumanized antibodies To determine BDG17.038, BDG17.043, BDG17.053, BDG17.054, BDG17.067, BDG17.066 and BDG 17.069 binding kinetics and affinity to hIL-2 and cynomolgus monkey IL-2 ), the clones were reformatted t0 IgG sed and d; Subsequently the antibodies were analyzed by SPR on a BIAcore T200 using the GE capture antibody kit as described herein. As shown in Table 9 and FIGs. G and FIGs. 15A-15B the antibodies bind tightly both human and cynomolgus monkey IL-2 an the low double digit pM range.

Size Exclusion Chromatographypro?le and thermal stability ofthe humanized antibodies.

To test if the humanized IgGs 038, BDG17.043, BDG17.053, 054, BDG17.066, BDG17.067 and BDG 17.069 are folded tly and stable, the antibodies were subjected to size exclusion tography and Differential Scanning Fluorimetry (DSF) analysis as described above. The results summarized in FIGs. 16A-16G and Table 8 suggesting that these IgGs are produced as >95% non-aggregated species, have a SEC retention pro?le typical of a human IgG1 and thermal denaturation profile with Tonset 0f >=54.4OC and Tml >=69OC. An illustration of the receptor discrimination assay is presented in A, and the SPR results for the different clones is presented in FIGs. 17B-17G. To conclude the SPR, SEC and DSF experiments indicate that the humanized antibodies bind human and cynomolgus money IL-2 tightly, are folded correctly and are highly stable.

TABLE 9: Biophysical Properties of Select Improved Humanized Clones IgG Size exclusion SPR SPR informati chromatography Kinetics of binding human IL—2. . . . Kinetics of binding. . . .

GE superdex200 Cynomolgus monkey IL—2 se 10/300 Ka Kd KD Ka Kd KD (MS'I) (8'1) (M) (MS’I) (8’1) (M) 1.41E+ 2.00E—04 2.02E- Not Not Not 07 1 1 tested tested tested N/A 2.22E-04 4.82E— Not Not Not 1 1 tested tested tested 3.96E+ 03 6.43E- Not Not Not OC ml 07 1 1 tested tested tested 17.054 Humaniz 61.8 69.0 74.60 13.04 4.16 5.50E+ 7.00E-04 1.27E- Not Not Not ed IgG1 0C 0C C ml 07 11 tested tested tested Humaniz N/A 12.96 3.8 7.85E—04 5.15E— Not Not Not ed IgG1 ml 11 Tested Tested Tested reduced gamma binding (LALA mutation) Humaniz N/A 13.02 4.7 2.83E 3.98E— 1.40E- ed IgG1 +07 04 11 reduced gamma binding (LALA 1.10E- mutation) 3.72E—04 1 1 Humaniz N/A 13.07 4.98E 5.84E— 1.17E- ed IgG1 ml +07 04 11 reduced gamma binding (LALA 1.08E— mutation) 4.87E—04 1 1 EXAMPLE 3 This example provides disclosure on anti-IL-2 antibodies that speci?cally block the g of human IL-2 to IL-2 receptor CD25 and modulate the immune system in ViVO.

Anti-IL-2 antibodies that bind human IL-2 at an epitope that specifically block interaction between IL-2 and human CD25, have several implications. This allows for the binding of IL-2 antibody x to effector T cells and NK cells but prevents the binding of human IL-2 to non- immune cells expressing high levels of CD25 (e.g., lung endothelium and vascular endothelium) or to immune cells expressing the high affinity trimeric x (e.g., Treg cells and CD25+ short lived cytotoxic effector T cells). As a result, these anti-IL-2 dies are able to expand effector T cells and NK cells without significantly expanding regulatory T cells (See, and FIG.- 2).

Additionally, it has been previously shown that lung endothelial cells express CD25 and high levels of IL-2 in their presence would lead to pulmonary edema. Similar effect was ed for IL-2 induction of vascular leal cells. Thus, by targeting IL-2 away from CD25, the anti-IL-2 antibodies disclosed herein are also ed to reduce any IL-2 related pulmonary and vascular toxicity (FIGS. 3A-3B).

Receptor mination ofthe humanized IgG-hIL-Z Complex id="p-324" id="p-324" id="p-324"

[0324] BDG17.023-hIL-2 complex (anti-IL-2 antibody-human IL-2 complex) showed receptor binding discrimination, the BDG17.023-hIL-2 complex was found to bind CD122 but not CD25 which resulted in a specific immune system modulation outcome. To test if the zed antibodies have a similar effect, they were complexed with hlL-2 and tested for binding to CD122 and CD25 by SPR. The analysis was done in a similar n to BDG17.023 as described herein. As can be seen by the SPR traces in FIGS. 17B-17G when the humanized antibodies BDG17.038, BDG17.043, BDG17.053, 054, 066, BDG17.0067 or BDG17.069 are complexed with hIL-2, the complex bind CD122 but cannot bind CD25, indicating that these antibodies retained the binding discrimination properties of human rat chimera 023.

In vivo Characterization ofthe humanized dies id="p-326" id="p-326" id="p-326"

[0326] It has been hypothesized that blocking the CD25 binding epitope on IL-2 by high affinity antibodies allows for the binding of human IL-2 to effector T cells and NK cells but prevents the binding of human IL-2 to non-immune cells sing CD25 (e.g., lung endothelium and vascular endothelium) or cells expressing the trimeric complex (e.g., Treg cells, CD25+ effector T cells). To test this hypothesis in vivo, anti-IL-2 dies were pre-complexed with human IL-2 and administered to healthy C57BL/6 male mice as described herein (FIGS. 18A-B). As can be seen in FIGS. 18A and 18B the anti-IL-2 antibodies BDG17.043 and BDG17.054, were able to expand effector T cells, NKT cells and NK cell populations without signi?cantly expanding regulatory T cells (A-18B), likely due to their epitope specific properties. Additionally, the proliferation of MP CD8+ T cells and NKT is dependent on the administered dose of IgG-hIL-2 complex and is much more robust than the administered e control with hIL2, suggesting that BDG17.043HL-2 and BDG17.054/IL-2 are actively promoting the CD122/CD132 dimer activation pathway while g the CD25/CD122/CD132 trimeric pathway (Figs. 19A-19B).

BDG17.043 and BDG17.054 complexed with IL-2 induced proliferation of MP CD8+ effector T cells and NK cells but do not promote icant expansion of tory T cells, suggesting that these antibody- 1L2 complexes have strong stimulatory effect on the immune system as opposed to other dies IL-2 xes like JES 6.1-IL-2 (Spangler JB, Tomala J, Luca VC, Jude KM, Dong S, Ring AM, Votavova P, Pepper M, Kovar M, Garcia KC. Antibodies to Interleukin- 2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms.

Immunity. 2015 May l9;42(5):815-25.) and Pfizer’s F5111.2-IL2 (Trotta E, Bessette PH, Silveria SL, et al. A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism. Nat Med. 2018;24(7)21005-1014.) that induce immune system anti-stimulatory effect by promoting proliferation of Tregs.) In the mouse study described above, the s were monitored daily for body weight loss and for non-specific clinical signs. When evaluating drug compounds in mice, a 20% percent body weight loss is ered to be an actionable item that requires ethical intervention. As shown in FIGS. 20A-B, mice administered with and 17.043HL-2 complex or 17.054?L-2 complex showed no or less than 10% percent of body weight loss at the end of the experiments. This effect was observed in all dose s including mice treated with the highest dose of 25 ug IgG/1.25ug IL-2 complex, indicating that the administered complexes are well tolerated.

Activity ofthe humanized antibodies in B16F10 syngeneic cancer model id="p-330" id="p-330" id="p-330"

[0330] Both viral nce and cancer therapy share the common need to expand the acquired T cell immune response for efficacy. The ability of anti-IL-2 antibodies to effectively activate immune response was tested in a B16F10 syngeneic ma model. C57BL/6 mice were inoculated with B16F10 melanoma and treated with 10ug anti-IL-2 antibody/lug hIL-2 complex or PBS control as described herein. As shown in A, all mice d with 043 or BDR17.054 complexed with IL-2 showed significant tumor growth inhibition 8 days post treatment that is much larger then treating the mice with Isotype control and hIL-2 (study day 17), likely due to robust and specific immune stimulation. onally, as can be seen in B the average transient weight loss in mice treated with the two anti-IL-2 antibodies is of 6.84%+/— 3.9% and —5.2%, indicating that in a setting of syngeneic B 16F10 tumor model the administered antibodyHL-2 complexes were well tolerated.

In summary, these studies show that anti-IL-2 antibodies (17.043 & 17.054) bind human IL- 2 with high affinity on a pre-defined epitope such that the antibodies completely prevent the interaction of IL-2 with its receptor CD25. Consequently, the antibody/IL-2 complex is directed to bind and activate the dimeric form of the IL-2R /CD132).

The dimeric receptor complex is found on effector cells. Analysis of the immune stimulating effect in viva demonstrated that IL-2 in the presence of anti-IL-2 clones 17.043 and 17.054, increases T-effector cell populations (IL-2 RBv binding and signaling) with no observed effect on regulatory T cells (IL-2 ROLB’Y binding and signaling). This demonstrated that the interaction of IL-2 with the dimeric IL-2 receptor resulted in non-toxic immune ation. Taken together, these data support the esis that an uman IL-2 dy that eres with the ability of the cytokine to bind CD25 ve cells could be used to treat oncologic patients to enhance immune responses that lead to immune response against cancer or in the case of COVID-19 infection can increased clearance of viral load. Additionally, these antibodies properties may prevent ILinduced pulmonary edema and possibly prevents lung tissue damage in SARS-CoV-2 infected lungs.

EXAMPLE 4 [033 3] This example provides a description of studies performed examining formulations of anti-IL- 2 dies.

Methods: Formulation analysis was done by incubating 30 mg/ml of L-2 clone BDG17.069 in four different formulations: 0 F1) 20 mM Histidine, 8% sucrose, 0.04% PS 80, pH5.5; 0 F2) 20 mM Histidine, 8% e, 0.04% PS 80, pH6.0; 0 F3) 20 mM Citrate, 8% e, 0.04% PS80, pH5.5; and 0 F4) 20 mM Histidine, 8% sucrose, 10 mM Methionine, 0.04% PS80, pH5.5. [033 5] The antibody was subjected to (1) incubation for one and two weeks at 40°C, (2) agitation of 300rpm at 25°C for three days, and(3) 3-5 cycles of Freeze/Thaw (F/T). At T=0 and post treatment the antibody was analyzed for appearance, size-exclusion chromatography-ultraperformance liquid chromatography (SEC-UPLC), pH, protein concentration, PI (Capillary isoelectric focusing -cIEF), subvisible particles (Micro ?ow g -MFI) and Tm (DSC).

Results: The tables presented as FIGS. 22A-22G, show the results of analyzing the different antibody formulations.

As can be seen in tables provided in FIGS. 22A-22G 069 formulated in F2, F3, F4 showed no apparent change in concentration, nor change in appearance is detected after one and two weeks of incubation at 40°C. Analysis of sub-visual particles by MFI trate that in BDG17.069 formulated in F1, F2, or F4 shows no formation of particles >=25uM and only minor change in formation of les >=10uM (B). SEC-UPLC analysis revealed only minor increase in small MW species for all four formulation conditions. Additionally, caliper-SDS analysis demonstrated minimal changes for BDG 17.069 formulated in F2 and F4, and analysis by cIEF displayed relatively small changes for BDG17.069 formulated in F4. However, BDG17.069 formulated in F3 ted substantial increase in acidic percentage at 40°C after 2 weeks of incubation (C).

While BDG17.069 formulated in F2 and in F3 exhibited slight particle formation post agitation (D), and after 5 cycles of Freeze/Thaw, BDG17.069 formulated in F4 and F1 did not show significant s to its ance, pH, concentration or any >5uM sub visible particles formation (F).

Taken together these experiments demonstrate that in formulations F1, F2, F3 and F4, BDG17.069 has good stability performance after agitation, freeze-thaw stresses and a very te aggregate percentage after incubation at 400C for 2 weeks; taking all stress conditions into t it is apparent that BDG 17.069 is most stable when formulated in 20 mM Histidine, 8% sucrose, 10 mM Methionine, 0.04% PS80, pH5.5 (F4).

EXAMPLE 5 This example provides a description of studies demonstrating the safety and efficacy of e specific anti-hIL-2 antibodies that are designed to enhance the immune response to infectious agents such as SARS-CoV-2 by specifically activating effector T and NK cells while reducing the binding of IL-2 to regulatory T cells and lung elium.

PRE—CLINICAL STUDIES The following inical studies can be done in 2 phases: phase 1 is to demonstrate the safety of the compound as a single intravenous dose in healthy volunteers; phase 2 is to demonstrate safety of multidoses in a virally infected animal model.

As disclosed herein, the anti-IL-2 antibodies specifically bind to human IL-2 and do not bind to mouse IL-2. Although mouse IL-2 receptors can bind human IL-2, allowing one to load hIL-2 into a complex and use a mouse model, normal mice are not desirable as they would subsequently begin to produce high levels of mouse IL-2. Secreted mIL-2 would therefore be available to bind receptors in the lung to induce edema, thereby confounding the safety interpretation. Therefore, animal models that allow either use of human IL-2 or for which dy cross reactivity with endogenous IL-2 does occur are preferred models. For mouse models, several s are available. First, to investigate whether the antibody or AbHLZ complex has any ental effect directly on CD25 expressing lung tissue, an IL-2 knock-out mouse is available. The primary limitation of this model is the lack of any subsequent amplification of the immune response and any additional immune elicited cytokines. A second option is to use healthy C57BL/6 or Bale mice which have been depleted of mouse IL-2 using a neutralizing antibody. However, optimization of this model will still be needed. A third option is to use CD34+ human cord blood cells transferred into the L mouse to generate a mouse expressing a human immune system. The advantage is that a near full human immune system (albeit with some limitations) is expressed, particularly with all T cell and NK cell populations. This model is also conducive for use as an acute viral infection model as well as a model system in oncology research.

Based data presented herein, since 17.067 and 17.069 demonstrated robust affinity s cynomolgus monkey IL-2 and based on the high homology between hIL-2 and cIL-2 are likely to ted Ab-IL2 complex receptor discrimination for both ; e safety models can be ted.

Healthy cynomolgus monkeys can be tested in a single ascending dose test. It has been demonstrated that cynomolgus monkeys can be infected by SARS-CoV-l and rhesus monkeys have been shown to be infected by SARS-CoV-Z. In both situations, a mild case of lung edema was observed, similar to what is observed in mild human ion. As such, these monkey models would allow for examination of both the safety and efficacy of the anti-IL-2 antibodies/IL-2 complexes.

Dose escalation experiments can be done in healthy animals (e.g. humanized mice, cynomolgus monkey etc) as follows: Supporting First in Man (FIM) y a. IL-2 KO mouse dose escalation; and/or b. mIL2 antibody depleted ntibody; and/or c. CD34+ SCT humanized mouse (+/— 1L2); d. Healthy cynomolgus or rhesus single dose escalation; e. Healthy cynomolgus or rhesus multi dose (done concurrently With FIM single ing dose).

Supporting patient dosing (phase lb or 2) a. Mouse (b or c above and/or hACE2 transgenic) viral loaded (acute ?u infection model or coronavirus respectively). b. If available, lgus (SARS-CoV) or rhesus (SARS-CoV-2) coronavirus acute infection model.

CLINICAL TRIALS id="p-348" id="p-348" id="p-348"

[0348] Part 1) A dose escalation trial in healthy volunteers can be done to identify the maximum tolerated dose. Up to eight cohorts of 10 individuals (8 test, 2 placebo) can be done using an appropriate interval n doses to all for safety determinations.

Part 2) Using the doses determined in Part 1, recently symptomatic oV-2 positive patients With mild symptoms can be treated and monitored for safety and efficacy. Efficacy can be determined by decrease in time to viral clearance and a decrease in the signs and symptoms of respiratory infection. Exploratory endpoints can include changes in eral blood immune cell populations and activation states.

CHEMISTRY, CTURING AND CONTROLS Cell line, drug substance (DS), and drug product (DP) can be done in a GMP qualified cturer under GMP guidance (e.g., Wuxi Biologics). An accelerated material production can be done to have material in 6 months ready for an IND. All, den, viral clearance, viral load and host cell proteins can be examined and reduced to specifications as determined by current cturing guidelines to ensure product safety. DP can be formulated for intravenous administration. Stability testing can be done concurrently to ensure product quality over time.

Claims (34)

1. An isolated anti-IL-2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, said VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein said CDRs have the amino acid sequences of the HCDR1 comprises the amino acid sequence of SEQ ID NO:62, the HCDR2 comprises the amino acid sequence of SEQ ID NO:63, the HCDR3 comprises the amino acid sequence of SEQ ID NO:64, the LCDR1 comprises the amino acid sequence of SEQ ID NO:65, the LCDR2 comprises the amino acid sequence of SEQ ID NO:66, the LCDR3 comprises the amino acid sequence of SEQ ID NO:67.

2. The antibody of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO:26, the VL comprises the amino acid sequence of SEQ ID NO:27.

3. The antibody of claim 1 or claim 2, wherein the antibody comprises an IgG, IgA, IgM, IgE, IgD, a Fv, a scFv, a Fab, a F(ab')2, a minibody, a diabody, or a triabody.

4. The dy of any one of claims 1-3, wherein said antibody comprises a heavy chain sing a mutation that reduces binding to Fc? or.

5. The antibody of claim 4, said antibody comprising a heavy chain ce and a light chain sequence, said heavy chain sequence set forth in SEQ ID NO: 72 and said light chain sequence set forth in SEQ ID NO: 73.

6. A ition comprising the dy of any one of claims 1-5 and a pharmaceutically acceptable carrier.

7. The composition of claim 6, wherein the composition is formulated to be at a pH between 20134079_1 (GHMatters) P119595.NZ about pH 5.0 - 6.0 and comprises a buffer ed from a histidine buffer and a citrate buffer.

8. The composition of claim 7, said composition further comprising at least one of sucrose, methionine, or PS80, or any combination thereof.

9. The composition of any one of claims 6-8, said composition further comprising IL-2.

10. An isolated polynucleotide sequence encoding the heavy chain variable region (VH) of an anti-IL-2 dy, wherein the VH amino acid sequence is set forth in SEQ ID NO:26, and the light chain variable region (VL) of an anti-IL-2 antibody, wherein the VL amino acid sequence is set forth in SEQ ID NO: 27.

11. A vector sing the polynucleotide sequence of claim 10.

12. A non-human or isolated host cell comprising the vector of claim 11.

13. An isolated polynucleotide sequence encoding an anti-IL-2 scFv, wherein the polynucleotide sequence is set forth in SEQ ID NO: 35.

14. A vector comprising the polynucleotide sequence of claim 13.

15. A non-human or ed host cell comprising the vector of claim 14.

16. Use of the anti-IL-2 antibody of any one of claims 1-5 in the manufacture of a medicament for promoting differential growth of subsets of immune cells and decreasing undesirable s caused by IL-2 in a subject having a disease or a condition, wherein said anti-IL-2 antibody is to be administered to treat said e or condition in said subject.

17. Use of the L-2 antibody of any one of claims 1-5 in the manufacture of a ment for treating a disease or a condition in a subject, wherein said medicament is to be administered to promote differential growth of subsets of immune cells and to decrease 20134079_1 (GHMatters) P119595.NZ undesirable effects caused by IL-2 to treat said disease or condition in said subject.

18. The use of claim 16 or 17, wherein said medicament comprises the anti-IL-2 antibody and IL-2, or the anti-IL-2 antibody complexed with IL-2.

19. The use of any one of claims 16-18, wherein the disease comprises a viral infection, a bacterial infection, or a .

20. The use of any one of claims 16-19, wherein said condition comprises a genetic predisposition that increases likelihood of cancer in said subject.

21. The use of claim 20, wherein said genetic predisposition comprises a change in expression or activity of a gene product, said gene comprising a tumor suppressor gene or a mismatch repair (MMR) gene, or a combination thereof.

22. The use of claim 20, wherein said genetic predisposition comprises a change in expression or activity of a gene product, said gene comprising BRCA1, BRAC2, MLH1, MSH2, MSH6, PMS1, PMS2, TP53, or CHEK2, or a combination thereof.

23. The use of any one of claims 16-22, wherein the immune cells comprise one or more of naïve T cells, memory T cells, CD8+ T cells, NK cells, or l Killer T cells.

24. The use of any one of claims 16-23, wherein said undesirable effect caused by IL-2 ses one or more of activation of regulatory T cells, apoptosis of CD25+ T effector cells, IL-2 induced pulmonary edema, IL-2 induced pneumonia, or ILinduced ar leakage.

25. The use of any one of claims 16-24, wherein said anti-IL-2 dy inhibits IL-2 g to CD25. 20134079_1 (GHMatters) P119595.NZ

26. The use of any one of claims 16-25, wherein said subject is to be further treated with one or more immune checkpoint inhibitors targeting one or more immune checkpoints.

27. The use of claim 26, wherein said subject is to be treated with said immune checkpoint inhibitors concurrently, before, or after treatment with said anti-IL-2 antibody.

28. The use of claim 26 or claim 27, n said immune checkpoint comprises PD-1, PDL1 , CTLA-4, TIGIT, TIM-3, B7-H3, CD73, LAG3, CD27, CD70, 4-1BB, GITR, OX40, lpha (CD47), CD39, ILDR2, VISTA, BTLA, or VTCN-1, or a combination thereof.

29. Use of the anti-IL-2 antibody of any one of claims 1-5 in the manufacture of a medicament for protecting a subject against pulmonary edema and lung tissue damage caused by SARSCoV-2 or IL-2.

30. The use of claim 29, wherein said medicament ses the anti-IL-2 antibody and IL-2, or comprises an anti-IL-2 antibody complexed with IL-2.

31. The use of claim 29 or claim 30, wherein said subject has a ed immune system.

32. The use of any one of claims 29-31, wherein said t has a genetic predisposition that increases likelihood of cancer in said t.

33. The use of claim 32, wherein said genetic predisposition comprises a change in expression or activity of a gene product, said gene comprising a tumor suppressor gene or a mismatch repair (MMR) gene, or a ation thereof.

34. The use of claim 32, wherein said genetic predisposition comprises a change in expression or activity of a gene product, said gene comprising BRCA1, BRAC2, MLH1, MSH2, MSH6, PMS1, PMS2, TP53, or CHEK2, or a combination thereof. 20134079_1 (GHMatters) P119595.NZ ”wmmo? +++ .1. N?: mag NNEQ mmwQQ WQ somwcmgxm comma .ucoo mm» we £91 Eowmagmmocsggmv Emwwwm Emmcmgumg NR: wmg??m SO .0505 commgmmxm E04 NEE?» +++ + MEX/Ewan“ w‘xum?ummm mm; mg Amam?amswocmggs M mg wmo me0 SEQ awmnm $§§m$ A3 av 3 3% 3mm go.“ §x mmxmmm?ou 3% “$803” wt: m mggmm 933mm mcmvgm 3% “Nags 3333 m mmgm WNEQ $50 xmaEau gm“:K EEK.» mEEEE ® _ “NJ” 5388 3 mmxm “8 $3335 mc?c? 3“ 5&va Egg SUBSTITUTE SHEET (RULE 26) $8? $8 +++ ++ $2 .. 5.8%}, mzmu mama +++ + + mgamumwcm wmo mmmm wwwmo xz wxz waQU 953 mmwmm?oncm mmmu mmwmm mmwmu mwmgmhmgw mmm?gmgm gaxmiwuq mommg?m mmmm mEBQBEQEQ Eugmm mg?gm g mg?ggm mm: .. 14% w gag» cm?whmmm mmmm 305ch © wmwmu Nam u< - 353. E. 32ng mmmmuéumx?ow? +++ +++ +++ + + 30 mme NNEU >553QO meU NNEO mmwmm 35% 3 §gm 53mm mc?cmm ?ggm NNEU®N 350$ Em?wm?w - mcngm mmm?gm Maggi“. 2% 3E3EE5§< ma?a mmsmmau ”$89 3+ + + $0 $30 $30 $50 SUBSTITUTE SHEET (RULE 26) mammmamm mm?w “xmm Emma mw?ugcmg 33$ Egmm mmwcmwom wmugm? fmcogma S ma: 1 CQEEEEE: @ [ III QM mm?m 3%? comcgma comcgma? €338on QNQN ghmw @993 om mEmwm magma“ mhméngoz >5:ng €385: 3% 33% E \Cmcogmna > 3-;ch majrcEm 330mg E3 m REESE Tat/om commmmhmohm SUBSTITUTE SHEET (RULE 26) {3325 Binding Site Biackage immune Amivaiiam Shgrt Eived CD8 cytomxéc T-cekés (3925 +++ CD122 +++ (38132 M CD8 MP s NK cans Reguiamry T-ceifs NKT ceiis (3325 +++ C025 .. CD122"? CD122 +++ (301324" C3132 ++ “:2 Taxicity Preventian Enhanced iL—2 secreiéan L & E Activated ung vascu ar Endotheliai Ceiis T_Ce”S CD25 +++ (30122 + CD132 + Fi?-35.. 33 SUBSTITUTE SHEET (RULE 26) | | | | _>_C 2: _>_: