Title of Invention: Recombinant Antibodies To Programmed

Death 1 (PD-1) And Uses Therefor

Technical Field

[1 ] The present invention relates to antibodies that bind to programmed cell

death 1 (PD-1 ) and particularly therapeutic and diagnostic methods of using those antibodies. The present invention belongs to the field of biotechnology.

Background Art

[2] In general, a need exists to provide safe and effective therapeutic methods for immune disorders such as, for example, autoimmune diseases, inflammatory disorders, allergies, transplant rejection, cancer, immune deficiency, and other immune system-related disorders. Modulation of the immune responses involved in these disorders can be accomplished by manipulation of the PD-1 pathway.

[3] An adaptive immune response involves activation, selection, and clonal

proliferation of two major classes of lymphocytes termed T cells and B cells. After encountering an antigen, T cells proliferate and differentiate into antigen-specific effector cells, while B cells proliferate and differentiate into antibody-secreting cells.

[4] T cell activation is a multi-step process requiring several signaling events between the T cell and an antigen-presenting cell (APC). For T cell activation to occur, two types of signals must be delivered to a resting T cell. The first type is mediated by the antigen-specific T cell receptor (TcR), and confers specificity to the immune response. The second, costimulatory, type regulates the magnitude of the response and is delivered through accessory receptors on the T cell.

[5] A primary costimulatory signal is delivered through the activating CD28

receptor upon engagement of its ligands B7-1 or B7-2. In contrast, engagement of the inhibitory CTLA-4 receptor by the same B7-1 or B7-2 ligands results in attenuation of T cell response. Thus, CTLA-4 signals antagonize costimulation mediated by CD28. At high antigen concentrations, CD28 costimulation overrides the CTLA-4 inhibitory effect. Temporal regulation of the CD28 and CTLA-4 expression maintains a balance between activating and inhibitory signals and ensures the development of an effective immune response, while safeguarding against the development of autoimmunity.

[6] Molecular homologues of CD28 and CTLA-4 and their B-7 like ligands have been recently identified. ICOS is a CD28-like costimulatory receptor. PD-1 (Programmed Death 1 or CD279) is an inhibitory receptor and a counterpart of CTLA-4. This disclosure relates to modulation of immune responses mediated by the PD-1 receptor.

[7] PD-1 is a 50-55 kDa type I transmembrane receptor that was originally

identified in a T cell line undergoing activation-induced apoptosis. PD-1 is expressed on activated T cells, B cells, and macrophages. The ligands for PD-1 are the B7 family members PD-L1 (B7-H1 ) and PD-L2 (B7-DC).

[8] PD-1 is a member of the immunoglobulin (Ig) superfamily that contains a single Ig V-like domain in its extracellular region. The PD-1 cytoplasmic domain contains two tyrosines, with the most membrane-proximal tyrosine located within an ITIM (immuno-receptor tyrosine-based inhibitory motif). The presence of an ITIM on PD-1 indicates that this molecule functions to attenuate antigen receptor signaling by recruitment of cytoplasmic

phosphatases. Human and murine PD-1 proteins share about 60% amino acid identity with conservation of four potential N-glycosylation sites, and residues that define the Ig-V domain.

[9] Experimental data implicates the interactions of PD-1 with its ligands in down regulation of central and peripheral immune responses. In particular, proliferation in wild-type T cells but not in PD-1 -deficient T cells is inhibited in the presence of PD-L1. Additionally, PD-1 -deficient mice exhibit an autoimmune phenotype. PD-1 deficiency in the C57BL/6 mice results in chronic progressive lupus-like glomerulonephritis and arthritis. In Balb/c mice, PD-1 deficiency leads to severe cardiomyopathy due to the presence of heart- tissue-specific self-reacting antibodies.

[10] Since PD-1 plays an important role in autoimmunity, tumor immunity and infectious immunity, it is an ideal target for immunotherapy. Blocking PD-1 with antagonists, including monoclonal antibodies, has been studied in treatments of cancer and chronic viral infections (Sheridan 2012, Nature Biotechnology 30: 729-730).

[1 1 ] Monoclonal antibodies to PD-1 are known in the art and have been

described, for example, in US Patent/Publication Nos.

US8008449, US8779105, US9084776, US9358289, US9387247,

US20090217401 , US20130133091 ,US20140212422, US20140294852, US20140328833, US20140348743, US20150165025, and WO Patent/ Publication Nos. WO2006121 168, WO20091 154335, WO2012145493, WO2013014668, WO200910161 1 , EP2262837, and EP2504028.

Summary of Invention

The present invention relates to anti-PD-1 antibodies and methods of using the same.

[12] The present disclosure provides antibodies that can act as agonists and/or antagonists of PD-1 , thereby modulating immune responses regulated by PD- 1. The disclosure further provides anti-PD-1 antibodies that comprise novel antigen-binding fragments. Anti-PD-1 antibodies of the invention are capable of :

(A) Specifically binding to PD-1 , including human PD-1 ;

(B) Competeting PD-1 binding to Nivolumab;

(C) Blocking PD-1 interactions with its natural ligand(s); or

(D) Performing both functions. [13] In particular embodiments, the four antibodies dervied and defined from rabbit and further be humanized comprise a heavy chain variable region (V _H ) and/or a light chain variable region (VL) and their Complementarity

Determining Region (CDR) as summaried in Table below:

Light Chian (V _L ) 55 DNA

Light Chian (V _L ) 56 Protein

Heavy Chian (V _H ) 57 DNA

Heavy Chian (V _H ) 58 Protein

Abs-r-925

Light Chian (V _L ) 59 DNA

Light Chian (V _L ) 60 Protein

Heavy Chian (V _H ) 61 DNA

Heavy Chian (V _H ) 62 Protein

Abs-r-926

Light Chian (V _L ) 63 DNA

Light Chian (V _L ) 64 Protein

Part D. A full variable sequence of humanized PD-1 antibodies

Sequence

Antibody Variable Region SEQ ID NO

Type

Heavy Chian (V _H ) 65 DNA

Heavy Chian (V _H ) 66 Protein

Abs-h-923

Light Chian (V _L ) 67 DNA

Light Chian (V _L ) 68 Protein

Heavy Chian (V _H ) 69 DNA

Heavy Chian (V _H ) 70 Protein

Abs-h-924

Light Chian (V _L ) 71 DNA

Light Chian (V _L ) 72 Protein

Heavy Chian (V _H ) 73 DNA

Heavy Chian (V _H ) 74 Protein

Abs-h-925

Light Chian (V _L ) 75 DNA

Light Chian (V _L ) 76 Protein

Heavy Chian (V _H ) 77 DNA

Heavy Chian (V _H ) 78 Protein

Abs-h-926

Light Chian (V _L ) 79 DNA

Light Chian (V _L ) 80 Protein

Description of Embodiments

[14] In general, the present invention provides rabbit antibodies and antigen- binding fragments thereof that bind to PD-1 , specifically in which they are recombinant antibody and also humanized antibodies.

[ 5] In one aspect, the invention provides an rabbit antibody and antigen-binding fragments which specifically binds to human PD-1 , and comprising a heavy chain variable region (H-CVR) selected from the group consisting of SEQ ID NOs: 1 - 3 (H-CDR1 , H-CDR2 and H-CDR3) and/or a light chain variable region (L-CVR) selected from the group consisting of SEQ ID NOs:13-15 (L-CDR1 , L-CDR2 and L-CDR3).

[16] In other aspect, the invention provides an rabbit antibody and antigen- binding fragments which specifically binds human PD-1 , and comprising a heavy chain variable region (H-CVR) selected from the group consisting of SEQ ID NOs:4-6 (H-CDR1 , H-CDR2 and H-CDR3) and/or a light chain variable region (L-CVR) selected from the group consisting of SEQ ID NOs: 16-18 (L-CDR1 , L-CDR2 and L-CDR3).

[17] In another aspect, the invention provides an rabbit antibody and antigen- binding fragments which specifically binds human PD-1 , and comprising a heavy chain variable region (H-CVR) selected from the group consisting of SEQ ID NOs:7-9 (H-CDR1 , H-CDR2 and H-CDR3) and/or a light chain variable region (L-CVR) selected from the group consisting of SEQ ID NOs: 19-21 (L-CDR1 , L-CDR2 and L-CDR3).

[18] in other aspect, the invention provides an rabbit antibodies and antigen- binding fragments which specifically bind human PD-1 , and comprising a heavy chain variable region (H-CVR) selected from the group consisting of SEQ ID NOs: 1 1 -12 (H-CDR1 , H-CDR2 and H-CDR3) and/or a light chain variable region (L-CVR) selected from the group consisting of SEQ ID NOs:22-24 (L-CDR1 , L-CDR2 and L-CDR3). [19] Preferably, the rabbit anti-PD-1 antibodies of the invention are selected from Abs-r-923, Abs-r-924, Abs-r-925 and Abs-r-926.

[20] In a preferred embodiments, the present invention provides an anti-PD-1 antibodies or antigen-binding fragments which are claimed as rabbit antibodies or fragments.

[21 ] In a further preferred embodiments, the present invention provides a rabbit antibodies or fragments which comprise a heavy chain variable region (H-CVR) further containing rabbit IgG or its variant with heavy chain FR region.

[22] In a further preferred embodiments, the present invention provides an rabbit antibodies or fragments which further contain rabbit IgG or its variant with heavy chain constant region.

[23] In a preferred embodiments, the present invention provides a rabbit

antibodies or fragments which comprise a light chain variable region (L-CVR) further containing rabbit k chain or its variant with light chain FR region.

[24] In a further preferred embodiments, the present invention provides an rabbit antibodies or fragments which further contain rabbit IgG or its variant with light chain constant region.

[25] In a preferred embodiments, the present invention provides an anti-PD-1 antibodies or antigen-binding fragments which comprise chimeric antibody or fragments.

[26] In a further preferred embodiments, the present invention provides an

chimeric antibody or fragments, and comprising a heavy chain variable region (H-CVR) from SEQ ID NOs: 50, 54, 58 and 62.

[27] in a further preferred embodiments, the present invention provides an

chimeric antibody or fragments, and comprising a light chain variable region (L-CVR) from SEQ ID NOs: 52, 56, 60 and 64.

[28] in one aspect, the invention provides an humanized antibody or antigen- binding fragments which specifically binds to human PD-1 , and comprising a heavy chain variable region (H-CVR) selected from the group consisting of SEQ ID NOs:25, 26 and 27 (H-CDR1 , H-CDR2 and H-CDR3) and/or their variants, and/or a light chain variable region (L-CVR) selected from the group consisting of SEQ ID NOs:37, 38 and 39(L-CDR1 , L-CDR2 and L-CDR3) or their variants.

[29] In other aspect, the invention provides an humanized antibody or antigen- binding fragments which specifically binds human PD-1 , and comprising a heavy chain variable region (H-CVR) selected from the group consisting of SEQ ID NOs: 28, 29 and 30 (H-CDR1 , H-CDR2 and H-CDR3) and/or their variants, and/or a light chain variable region (L-CVR) selected from the group consisting of SEQ ID NOs: 40, 41 and 42 (L-CDR1 , L-CDR2 and L-CDR3).

[30] In other aspect, the invention provides an humanized antibody or antigen- binding fragments which specifically binds human PD-1 , and comprising a heavy chain variable region (H-CVR) selected from the group consisting of SEQ ID NOs: 31 , 32 and 33 (H-CDR1 , H-CDR2 and H-CDR3) and/or their variants, and/or a light chain variable region (L-CVR) selected from the group consisting of SEQ ID NOs: 43, 44 and 45 (L-CDR1 , L-CDR2 and L-CDR3).

[31 ] In other aspect, the invention provides an humanized antibody or antigen- binding fragments which specifically binds human PD-1 , and comprising a heavy chain variable region (H-CVR) selected from the group consisting of SEQ ID NOs: 34, 35 and 36 (H-CDR1 , H-CDR2 and H-CDR3) and/or their variants, and/or a light chain variable region (L-CVR) selected from the group consisting of SEQ ID NOs: 46, 47 and 48 (L-CDR1 , L-CDR2 and L-CDR3).

[32] Preferably, the humanized anti-PD-1 antibodies of the invention are

selected from Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926.

[33] In a preferred embodiments, the present invention provides an anti-PD-1 antibodies or antigen-binding fragments which are claimed as humanized antibodies or fragments. [34] In a further preferred embodiments, the present invention provides an humanized antibodies or fragments which comprise a heavy chain variable region (H-CVR) further containing human lgG1 , lgG2, lgG3, lgG4 and/or a heavy chain FR region variant,

[35] In a further preferred embodiments, the present invention provides an

antibodies or fragments which further contain humanized antibody and/or a heavy chain constant region.

[36] In a preferred embodiments, the present invention provides an humanized antibodies or fragments which comprise a light chain variable region (L-CVR) further containing human k , λ chain or a light chain FR region variant.

[37] In a further preferred embodiments, the present invention provides an

humanized antibodies or fragments which further contain human IgG and/or a light chain constant region variant.

[38] In a preferred embodiments, the present invention provides an anti-PD-1 antibodies or antigen-binding fragments which comprise chimeric antibody or fragments, further containing human lgG1 , lgG2, lgG3, lgG4 and/or a heavy chain constant variant, preferred human lgG1 or lgG4.

[39] In a further preferred embodiments, the present invention provides an

chimeric antibody or fragments, and comprising a heavy chain variable region (H-CVR) from SEQ ID NOs: 66, 70, 74 and 78.

[40] In a further preferred embodiments, the present invention provides an

chimeric antibody or fragments, and comprising a light chain variable region (L-CVR) from SEQ ID NOs: 68, 72, 76 and 80.

[41 ] In some embodiments, the present invention provides an anti-PD-1 antibody or fragments, consisting of Fab, Fv, single chain variable fragment (scFv), F(ab') ₂ and diabody.

[42] In another aspect, the present invention provides an isolated nucleic acids, DNA molecules that encode any of the antibodies described herein. [43] In a further aspect, the present invention provides an isolated polynucleotide composition, comprising: an encoder of the present invention, the anti-PD-1 polynucleotide of the light chain of antibody or a functional fragment and the anti-PD-1 polynucleotide of the heavy chain of antibody or functional fragment thereof.

[44] In another aspect, antibody or antigen binding portion of the invention can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand receptor) to generate at least two different the bispecific binding sites or target molecules. Antibodies of the invention can in fact be derivatized or linked to more than one other functional molecule to generate more than two different binding sites and / or target molecule binding multispecific molecule; such multispecific molecules are also intended to be as used herein.

[45] In another aspect, the present invention provides an expression vector of comprising the nucleic acids thereof encoding the present invention.

[46] In a further aspect, the present invention provides an host cell of transfected expression vector.

[47] In another embodiments of the present invention is provided comprising host cells. In certain the host cell is selected from mammalian cells, more specifically, the mammalian cell is selected from Chinese hamster ovary cells.

[48] In some particular embodiments, according to the anti-PD-1 antibodies or functional fragments thereof of the present invention block the interaction and / or interact with PD-1 and PD-L2 and the PD-1 and PD-L1.

[49] In a further aspect, the present invention provides the use according to the anti-PD-1 antibody or a functional fragment of the present invention in the manufacture of a medicament for enhancing the immune response of T cells. In some embodiments, the enhanced immune response includes enhancement of T cell cytokines, preferably the cytokine comprises IL-2. [50] In a further aspect, the present invention provides the use according to the anti-PD-1 antibody or a functional fragment of the present invention for competitively binding to human PD-1 with Nivolumab antibody. In particular embodiments, the competitive binding includes blocking binding of antibody to PD-1 , preferably the antibody comprises Nivolumab.

[51 ] In another aspect, the present invention provides the use according to the anti-PD-1 antibody or a functional fragment of the present invention for the treatment or prevention of cancer or infectious diseases of the medicament.

[52] The present invention further provides a method of treating and preventing PD-1 mediated disease or disorder, or comprising pharmaceutical

compositions thereof; preferably wherein said disease is cancer, most preferably the breast cancer, lung cancer, stomach cancer, colon cancer, kidney cancer and melanoma.

Examples

[53] The following examples are provided to further explain and demonstrate some of the presently preferred embodiments and are not intended to limit the scope or content of the invention in any way.

Example-1 : Generation of rabbit anti-PD-1 Antibodies

[54] A. Immunization of rabbit with PD-1

Four female rabbits aged 4 months were used for immunizations. The each rabbit was subcutaneously primed with 50 g of own-made recombinant human PD-1 mixed well with complete Freund's adjuvant (Sigma- Aldrich,Cat#263810). Two to three weeks later, the rabbits were boosted with 25 ig of soluble PD-1 with incomplete Freund's adjuvant (Sigma- Aldrich, Cat#263910). The immune response was monitored by checked antiserum taken from rabbit's auricular vein blood. A serial dilutions of the antiserum from immunized animals were then screened for binding to the coated human PD-1 by ELISA. The same purified protein in PBS used for immunizations diluted to 1 g / ml, was coated overnight microtiter plate (Nunc MAXISORP), 10ΟμΙ / well incubated at 4°C overnight, then blocked with 200 l/well containing 5% fetal calf serum, PBS solution of 0.05% Tween 20. A serially diluted immunized sera were added to each well and incubated at room temperature for 1 hour. With PBS / Tween 20 solution washing the plate, horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Jackson Immunoresearch Labs, Cat #:1 1 1 -035-046) was incubated for 1 hour at room temperature. After the plate was washed with washing buffer, Ι ΟΟμΙ/weil of TMB substrate was added, then (Pierce, Cat # 34021 ) colored, stopped reaction and measured OD value at 450 nm.

[55] B. Screening of rabbit anti-PD-1 Antibodies for Binding to Human PD-1

According to titer comparison, the rabbits with anti-PD-1 high titer

immunoglobulin were chosen for isolation of rabbit PD-1 -specific B cell. Cells were interrogated for antigen-specific B cells, unstimulated or stimulated, using LPS (Invivogen) at a concentration of 20 ng/mL. The cells, unstimulated or stimulated, were then loaded into pre-coated and blocked plate as described below. 384 well plate coated with anti-rabbit IgG antibody (Jackson Immunoresearch, Cat #1 1 1 -005-008) was blocked with PBS solution containing 5% fetal calf serum and 0.05% Tween 20, then used to capture the antigen-specific B cells, contained the secreted output of the B cells. The antibodies secreted by the B cells in each well were then assayed for reactivity against human PD-1 , or an unrelated antigen, and also assayed for IgG reactivity. Mainly, following three combined approaches were further applied for efficiently screened rabbit anti-PD-1 antibodies with blocking function and high binding affinity.

(i) Screening of functional anti-PD-1 antibody by Fiuorometric icrovoiume Assay Technology, FMAT

Recombinant antibodies from cultured supernatants were also used in cell-based binding studies. HEK293 ceils were transfected with expression plasmids encoding the Ig heavy and light chains of rabbit anti-PD-1 antibodies. After 3-5 days, recombinant antibodies in the supernatants of transfected cells were harvested. Stable HEK293 cells expressing human

PD-1 or primary cells were used for the cell-based binding confirmation studies. Antibodies from cultured supernatants were assessed for binding to cell surface PD-1 using fiuorescentiy labeled anti-rabbit IgG antibody

(Jackson Immunoresearch, Cat #1 1 1 -095-046), Fluorescence microscopy studies revealed that a number of anti-PD-1 antibodies obtained from transfection supernatants bound to PD-1 expressed on the surface of

HEK293 cells. In this screening assay, own-made anti-PD-1 antibody and isotype control rabbit IgG were used as controls. The fig.1 A represents one of multiple 384-well plates detecting PD-1 antibodies from immunized rabbits that inhibit PD-1 function. Antibodies which show no inhibition are seen as signals above the plane of the graph. Antibodies that inhibit PD-1 function remain "flat" with the plane of the graph and represent the majority in FMAT screens. Fig.l B further summarize results of tests of binding of various rabbit anti-PD-1 antibodies to PD-1 expressed on the surface of HEK293 cells. Four antibodies with desired inhibition function and biding affinity were selected and confirmed by other assays below.

(ii) Screening of binding anti-PD-1 antibody by Enzyme-Linked

Immunosorbent Assay, ELISA

To begin the ELISA, 384-well plates were coated with human PD-1 , blocked with assay diluent buffer, and washed, prior to incubation with serial dilutions of supernatants from the cultured PD-1 -specific B-cells.

The samples were incubated for one hour, and then the ELISA plates were washed. The detection goat anti-rabbit antibody-conjugated with HRP in the same assay diluent buffer, was then added, the assay plate was washed, and substrate solution was added to the wells in the assay plate. After the enzyme reaction was stopped, colorimetric density at 450 nm was measured in a conventional plate reader. Over hundred of PD-1 - positive clones were screened by ELISA. Furthermore, the four of rabbit anti-PD-1 antibodies with specifically and strongly binding activity, named as Abs-r-923, Abs-r-924, Abs-r-925 and Abs-r-926 were defined and selected for characterizations (See in Fig.lC). Binding data showed that they did not react with other unrelated proteins as coated, but also did not have cross-reactivity with mouse PD-1 .

(iii) The interaction of PD-1 with PD-L1 blocked by selected anti-PD-1

antibody (See Example 3 below).

[56] C. Isolation of PD-1 Antibodies that bind to Human PD-1

Antibody-producing cells identified from screening above were used for isolated the genetic sequences encoding antibody heavy and light

chains The genes encoding the specific antibodies that recognize human PD-1 were retrieved using RT-PCR. Retrieved cells were placed into reverse transcription buffer, and the mRNA from each individual cell was reverse transcribed into cDNA. After generating these amplicons by standard nested polymerase chain reaction (PCR), these amplicons were subjected to direct sequencing. These DNA sequences were then bioinformatically filtered for sequence quality and organized into a sequence database for cladistics analysis, in order to identify how many unique antibody clades were isolated that recognize PD-1. According to the manufacturer's protocol, the amplified and selected sequences were cloned into HEK293 expression system for producing PD-1 antibody. Complete antibody variable regions comprising a pair of heavy (V _H ) and light chain variable regions (V _L ) were reformatted into plasmids with the proper elements for transient expression in mammalian cell lines, e.g., HEK293 using standard molecular biology techniques. For example, the variable heavy (SEQ ID NO:54) and variable light (SEQ ID NO:56) cDNA sequences were sub-cloned into the vector backbone.

Supernatants from transiently transfected mammalian cell lines were used to test for antibody expression, specificity and affinity. Various V _H and V _L were paired to yield more than hundred of clones, in which 10 (a to j clones) of them were selected and expressed in HEK293 cells and assayed for PD-1 functional inhibition assay (See Fig. l C). These analyses identified unique clades of sequences that recognize human PD-1. Selected sequences were sub-cloned into PCR 2.1 (LifeTech) for further propagation. The sequences of the individual heavy chain and light chain variable regions are shown in Figs. 3A, 4A, 5A and 6A (HCVR) and Figs. 3B, 4B, 5B and 6B (LCVR). The complementarity determining regions (CDRs) and framework regions (FRs) are indicated as well. Tables 1 , 2, 5, 6, 9,10, 13 and 14 list each H-CDR or L-CDR by clone name and corresponding sequence identifier, Abs- r-923, Abs-r-924, Abs-r-925 and Abs-r-926. They are also described in PD-1 antibody sequence listing.

Fig.1 D also showed that an example of an ELISA-based binding assay. The results represent an average of two experiments. According to PD-1 antibody ability to block the binding of PD-L1 , Abs-r-923, Abs-r-924, Abs-r-925 and Abs-r-926 clones are selected as a candidates for further development of a humanized antibody (See also in Example 2 below).

Example-2, Humanization of Rabbit Anti-PD-1 Antibodies

[57] Humanization of selected rabbit anti-PD-1 antibodies was performed in

order to reduce the apparent immunogenicity of the rabbit-based antibodies. Using antibody engineering information well-known in the art, and

conventional bioinformatics tools, amino acid sequences of certain rabbit anti- PD-1 antibodies of the invention were analyzed and compared against known human antibody sequences. Based on these analyses and comparisons, certain human sequences were chosen for conventional rabbit CDR grafting, and inclusion of suitable back mutations. In tests for binding to human PD-1 , these humanized antibodies were evaluated with respect to criteria such as function, affinity, avidity, binding kinetics, and biochemical behavior such as aggregation as well as expression levels.

A. Humanized heavy chain variable region sequence

With the known human germline immunoglobulin heavy chain sequences are compared, selected low immunogenicity human germline framework sequence to finalize humanization of four rabbit PD-1 antibodies, Abs-r- 923, Abs-r-924, Abs-r-925 and Abs-r-926. The DNA and amino acid sequences of the four rabbit recombinant monoclonal antibodies are shown in Figs. 3A, 4A, 5A and 6A, and also in SEQ ID NOs: 49, 50, 53, 54, 57, 58, 61 and 62, as well as further H-CDRs in Tables 1 , 5, 9 and 13 below. See these sequences also in PD-1 antibody sequence listing.

The DNA and amino acid sequences of the heavy chain variable region of humanized Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926 are shown in SEQ ID NOs: 65, 66, 69, 70, 73, 74, 77 and 78, and further H-CDR sequences in Tables 3, 7, 1 1 and 15. See these sequences also in PD-1 antibody sequence listing.

B. Humanized light chain variable region sequence

With the known human germline immunoglobulin light chain sequences are compared, selected low immunogenicity human germline framework sequence to finalize humanization of four rabbit PD-1 antibodies, Abs-r- 923, Abs-r-924, Abs-r-925 and Abs-r-926. The DNA and amino acid sequence of the light chain variable region of the four rabbit recombinant monoclonal antibodies are shown in Figs. 3B, 4B, 5B and 6B, and also in SEQ ID NOs: 51 , 52, 55, 56, 59, 60, 63 and 64, and further L-CDRs in Tables 2, 6, 10 and 14. See these sequences also in PD-1 antibody sequence listing.

The DNA and amino acid sequence of the light chain variable region of humanized Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926 are shown in SEQ ID NOs: 67, 68, 71 , 72, 75, 76, 79 and 80, and further L-CDRs in Tables 4, 8, 12 and 16. See these sequences also in PD-1 antibody sequence listing.

Example-3: Blocking of humanized PD-1 antibodies binding to PD-L1

[58] By blocking the PD-1 ligand binding manner of the present invention which is the humanized anti~PD-1 antibodies testing. Specifically, 100 μΙ of unlabeled human PD-L1 (R & D Systems, Cat # 156-B7-100) was coated at a

concentration of 1 pg/mL in PBS on a 96-well microtiter plates overnight at 4°C, Nonspecific binding sites were subsequently blocked using a 0.5% (w/v) solution of BSA and 0.05% (w/v) Tween 20 in PBS. In a blocking assay, a constant concentration of 1.5nM (0.5ug/ml) of human PD-1 protein was added to a serial dilutions of humanized anti-PD- antibodies or Nivoiumab or isotype control antibody. The antibody-protein complexes with 1.5 nM constant human PD-1 were transferred to microliter plates coated with human PD-L1. After incubating for 1 hour at RT, the wells were washed, and plate- bound human PD-1 was detected with an anti-human IgG Fc antibody conjugated with horseradish peroxidase (HRP) (Abeam, Cat#ab97225). After the plate was washed with washing buffer, 100 μΙ/weil of T B substrate was added, then colored, stopped reaction and measured OD value at 450 nm. As shown in Fig, 2, the humanized anti-PD-1 antibodies, Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926 still maintained their functions and specifically blocked the binding of PD-1 to its ligand PD-L1 , and significantly better than the blocking effect of Nivoiumab. Thus, the antibody of the present invention achieves a surprisingly higher for PD-1 blocking the binding of PD-L1.

Example 4. Construction and expression of a fully humanized PD-1 antibody

[59] Human igG4 heavy chain constant region fragment lgG4 Fc sequence and K light chain constant region fragment sequences were synthesized by the company IDT (Integrated DNA Technologies, Coralville, Iowa) respectively, then put to pcDNA3 vector backbone to pBA-H4 and pBA-Ck of the present invention, pBA-H4 (containing human igG4 heavy chain constant region lgG4Fc) and pBA-Ck (containing the human κ light chain constant region fragment) vector built by Bioabs company, using the CMV promoter pBA-H4 in VH and CH, puromycin resistance gene use PGK promoter, in pBA-Ck in VL use CMV promoter, neomycin resistance gene SV40 promoter. According to the protein sequence of the antibody heavy chain variable region sequence and light chain variable region encoding the heavy chain and light design chain variable region DNA sequences, and in accordance with optimizing expression in CHO cells, and further optimize the DNA sequences encoding the heavy and light chain variable region, wherein the encoding of the present invention is the humanized anti-PD-1 antibody heavy chain variable region DNA sequences are shown in SEQ ID NOs: 65 (Abs-h-923), 69 (Abs-h-924), 73 (Abs-h-925) and 77 (Abs-h-926), the present invention encoding the humanized anti-PD-1 antibody light chain variable region DNA sequence as SEQ ID NOs: 67 (Abs-h-923), 71 (Abs-h-924), 75 (Abs-h-925) and 79 (Abs-h- 926). All tested PD-1 antibodies were purified by protein A column containing suitable resin, MabSelect™ from GE Healthcare.

Example 5. Specificity and affinity of humanized PD-1 Antibodies

[60] The binding affnity of the anti-PD-1 antibodies Abs-h-923, Abs-h-924, Abs-h- 925 and Abs-h-926 were determined by surface plasmon resonance (SPR) using the Biacore™ technology. This technology allows the label-free determination of the microscopic rate constants for binding (k _a ) and

dissociation (k _d ) of a ligand to a receptor. It is therefore especially suited for characterizing the antibody-antigen interactions. Indirect binding of antibodies to the Biacore™ chip surface was done via an anti-human IgG antibody (GE Healthcare Bio-Sciences AB; Cat# BR-1008-39) 25 pg/ml in immobilization buffer (10mM Sodium acetate pH 5.0). Anti-PD-1 antibody in 2-fold increasing concentrations from 0.14 to 7.68 nM was diluted into assay buffer to measure affinity of Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926. The kinetic traces were evaluated with the Biacore™ T2000. The full set of these traces with increasing concentrations is taken together and is called a run. Two zero concentration samples (blank runs) were included in each analyte

concentration series to allow double-referencing during data evaluation. The kinetic rate constants for association (k _a ) and dissociation (k _d ) as well as the dissociation equilibrium constant (K _D ) were calculated. The affinity data of Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926 are shown in Table 17.

Example 6. Competitive Binding of Humanized PD-1 Antibodies with

Nivolumab [61 ] Competitive binding analysis was performed on humanized antibodies, Abs- h-923, Abs-h-924, Abs-h-925 and Abs-h-926 with Nivolumab, using ELISA- based assay. The assay was conducted essentially as follows. A different of concentration of PD-1 antibodies, Abs-h-923, Abs-h-924, Abs-h-925 and Abs- h-926 were pre-incubated with constant concentration, 50 ng/ml of Nivolumab or an isotype antibody (negative control) for 1 hour at 37°C. The mixtures were added into 96-well plate coated human PD-1 at concentration of 100 ng/well and further incubated for other hour, then washed. HRP-conjugated anti-human antibody was added and incubated. After the plate was washed with washing buffer, Ι ΟΟμΙ/well of TMB substrate was added, then (Pierce, Cat # 34021 ,) colored, stopped reaction and measured OD value at 450 nm.

All humanized antibodies competitively inhibited the binding of Nivolumab to PD-1. However, antibody Abs-h-923 performed weak competition binding to PD-1. Antibody Abs-h-926 had the highest competition activity compared to the other three. In contrast, isotype antibody did not competitively inhibit binding of Nivolumab to PD-1 at all. Representative results are shown in Fig.7.

The EC ₅ o of Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926 is estimated by determining concentration of Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h- 926 that exhibit roughly one-half maximal absorbance from the point at which the Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926 display saturation behavior (See Table 18).

Example 7. Humanized PD-1 Antibodies induced IL-2 Cytokine Secretion in MLR Assays

Increased secretion of IL-2 cytokine was observed in response to four antibodies Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926 in mixed lymphocyte reaction (MLR) assays. One commercial stage anti-PD-1 antibody, Nivolumab was used as benchmarks for this experiment. The MLR assay was performed using commercially available monocyte-derived dendritic cells as stimulator cells and purified CD4 ⁺ T lymphocytes as responder cells from a different healthy blood donor. Supernatants were collected at 2.5 days after beginning the assay. Cytokine secretion was measured in a capture sandwich immunoassay using MagPlex® microspheres (Luminex), according to the vendor's instructions.

Fig.8 summarized the results of an MLR assay performed on human PBMCs treated with the anti-PD-1 antibodies. The data indicated that treatment with humanized PD-1 antibodies, Abs-h-924, Abs-h-925 and Abs-h-926 resulted in significantly increased secretion of cytokine IL-2, in comparison with antibody Nivolumab or the lgG4 isotype control (Biolegend). Interestedly, antibody Abs-h-923 showed weaker induced IL-2 response than other three antibodies Abs-h-924, Abs-h-925 and Abs-h-926. These data indicate that some of the anti-PD-1 antibodies of the present invention, e.g., Abs-h-926, induce increased IL-2 cytokine release in a manner similar to Nivolumab, while one antibody, Abs-h-923, elicits physiological responses that are measurably different from the responses elicited by Nivolumab.

[62] [Table 1 ]

Table 1. A H-CD and full heavy chain variable sequences of rabbit

anti-PD-1 antibody Abs-r-923

[Table 2]

Table 2. A L-CDR and full light chain variable sequences of rabbit

[Table 3]

Table-3. A H-CDR and full heavy chain variable sequence of humanized

anti-PD-1 antibody Abs-h-923

[Table 4]

Table 4. A L-CDR and full light chain variable sequence of humanized

anti-PD-1 antibody Abs-h-923

[66] [Table 5]

Table 5. A H-CDR and full heavy chain variable sequences of rabbit

[Table 6]

Table 6. A L-CDR and full light chain variable sequences of rabbit

anti-PD-1 antibody Abs-r-924

[Table 7]

Table-7. A H-CDR and full heavy chain variable sequence of humanized

anti-PD-1 antibody Abs-h-924

[69] [Table 8]

Table 8. A L-CDR and full light chain variable sequence of humanized

[Table 9]

Table 9. A H-CDR and full heavy chain variable sequences of rabbit

anti-PD-1 antibody Abs-r-925

[Table 10]

Table-10. A L-CDR and full light chain variable sequences of rabbit

anti-PD-1 antibody Abs-r-925

[72] [Table 1 1 ]

Table 11. A H-CDR and full heavy chain variable sequence of humanized

[Table 12]

Table 12. A L-CDR and full light chain variable sequence of humanized

anti-PD-1 antibody Abs-h-925

[Table 13]

Table 13. A H-CDR and full heavy chain variable sequences of rabbit

anti-PD-1 antibody Abs-r-926

[75] [Table 14]

Table 14. A L-CDR and full light chain variable sequences of rabbit

[76] [Table 15]

Table 15. A H-CDR and full heavy chain variable sequence of humanized

anti-PD-1 antibody Abs-h-926

[77] [Table 16]

Table 16. A L-CDR and full light chain variable sequence of humanized

anti-PD-1 antibody Abs-h-926

[78] [Table 17]

Table 17. Binding affinity and kinetic rate constants

of humanized PD-1 antibodies

[79] [Table 18]

Table 18. EC50 of humanized antibodies binding

Antibody EC50 (nM)

Abs-h-923 0.41

Abs-h-924 0.14

Abs-h-925 0.15

Abs-h-926 0.18 Reference to Deposited Biological Material

[80] Not apply in this application. Sequence Listing Free Text

[81 ] SEE PD-1 ANTIBODY SEQUENCE LISTING FILE

Patent Literature

[82] PTL1 : WO2013173223A1

[83] PTL2: WO2015036394A1 ,

[84] PTL3: WO20151 12800A1,

[85] PTL4: WO20151 12900A1 ,

[86] PTL5: WO2016020856A3,

[87] PTL6: WO2016068801 A1

[88] PTL7: WO2016077397A3

[89] PTL8: WO2016106159A1

[90] PTL9: US 8735553 B1

Non Patent Literature

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[92] NPL2: Webster RM. The immune checkpoint inhibitors: where are we now? Nat Rev Drug Discov. 2014;13(12):883-4.

[93] NPL3: Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372(21 ):2018-28 [94] NPL4:Benson Jr DM, Bakan CE, Mishra A, Hofmeister CC, Efebera Y,

Becknell B, et al. The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-01 1 , a novel monoclonal anti- PD-1 antibody. Blood. 2010; 1 16(13):2286-94.

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Poddubskaya E, et al. Nivolumab versus docetaxel in advanced squamous- cell non-small-cell lung cancer. N Engl J Med. 2015;373(2): 123-35.

Brief Description of Drawings

[1 ] The invention can be more completely understood with reference to the

following drawings.

Fig.lA

[2] [fig.1A] shows summarizing data on specific and high-affinity binding of

selected rabbit anti-human-PD-1 recombinant antibodies by FMAT assay. It is further demonstrated in Examples 1 and 3 of present invention.

Fig.l B

[3] [fig.1 B] shows summarizing data on both PD-1 inhibition function and binding affinity of selected rabbit anti-human-PD-1 recombinant antibodies, Abs-r-923, Abs-r-924, Abs-r-925 and Abs-r-926. It is further demonstrated in Examples 1 and 3 of present invention.

Fig.lC

[4] [fig.1 C] shows summarizing data on binding specificity of selected rabbit anti- human-PD-1 recombinant antibodies, Abs-r-923, Abs-r-924, Abs-r-925 and Abs-r-926 by ELISA. It is further demonstrated in Examples 1 and 3 of present invention.

Fig.l D

[5] [fig.l D] shows summarizing data on inhibition activity of selected ten rabbit anti-human-PD-1 recombinant antibodies and picked four of them for humanization, Abs-r-923, Abs-r-924, Abs-r-925 and Abs-r-926. It is further demonstrated in Examples 1 and 3 of present invention. Fig.2

[6] [fig.2] shows summarizing data on PD-1 binding to PD-L1 inhibited by four humanized anti-human-PD-1 recombinant antibodies, Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926. It is further demonstrated in Examples 1 and 3 of present invention.

Fig.3A

[7] [fig.3A] shows DNA sequence (SEQ ID NO:49) and amino acid sequence (SEQ ID NO:50) of Heavy Chain Variable Regions (H-CVR) from rabbit anti- human-PD-1 recombinant antibody, Abs-r-923. Complementarity determining regions of H-CDR1 through H-CDR3 are also shown in Table 1. It is further demonstrated in Example 3A of present invention.

Fig.3B

[8] [fig.3B] shows DNA sequence (SEQ ID NO:51 ) and amino acid sequence (SEQ ID NO:52) of Light Chain Variable Regions (L-CVR) from rabbit anti- human-PD-1 recombinant antibody, Abs-r-923. Complementarity determining regions of L-CDR1 through L-CDR3 are also shown in Table 2. It is further demonstrated in Example 3B of present invention.

Fig.3C

[9] [fig.3C] shows DNA sequence (SEQ ID NO:65) and amino acid sequence (SEQ ID NO:66) of Heavy Chain Variable Regions (H-CVR) from humanized anti-human-PD-1 recombinant antibody, Abs-h-923. Complementarity determining regions of H-CDR1 through H-CDR3 are also shown in Table 3. It is further demonstrated in Example 3A of present invention.

Fig.3D

[10] [fig.3D] shows DNA sequence (SEQ ID NO:67) and amino acid sequence (SEQ ID NO:68) of Light Chain Variable Regions (L-CVR) from humanized anti-human-PD-1 recombinant antibody, Abs-h-923. Complementarity determining regions of L-CDR1 through L-CDR3 are also shown in Table 4. It is further demonstrated in Example 3B of present invention.

Fig.4 A

[1 1 ] [fig.4A] shows DNA sequence (SEQ ID NO:53) and amino acid sequence (SEQ ID NO:54) of Heavy Chain Variable Regions (H-CVR) from rabbit anti- human-PD-1 recombinant antibody, Abs-r-924. Complementarity determining regions of H-CDR1 through H-CDR3 are also shown in Table 5. It is further demonstrated in Example 3A of present invention.

Fig.4B

[12] [fig.4B] shows DNA sequence (SEQ ID NO:55) and amino acid sequence (SEQ ID NO:56) of Light Chain Variable Regions (L-CVR) from rabbit anti- human-PD-1 recombinant antibody, Abs-r-924. Complementarity determining regions of L-CDR1 through L-CDR3 are also shown in Table 6. It is further demonstrated in Example 3B of present invention.

Fig.4C

[13] [fig.4C] shows DNA sequence (SEQ ID NO:69) and amino acid sequence (SEQ ID NO:70) of Heavy Chain Variable Regions (H-CVR) from humanized anti-human-PD-1 recombinant antibody, Abs-h-924. Complementarity determining regions of H-CDR1 through H-CDR3 are also shown in Table 7. It is further demonstrated in Example 3A of present invention.

Fig.4D

[14] [fig.4D] shows DNA sequence (SEQ ID NO:71 ) and amino acid sequence (SEQ ID NO:72) of Light Chain Variable Regions (L-CVR) from humanized anti-human-PD-1 recombinant antibody, Abs-h-924. Complementarity determining regions of L-CDR1 through L-CDR3 are also shown in Table 8. It is further demonstrated in Example 3B of present invention. Fig.5A

[15] [fig.SA] shows DNA sequence (SEQ ID NO:57) and amino acid sequence (SEQ ID NO:58) of Heavy Chain Variable Regions (H-CVR) from rabbit anti- human-PD-1 recombinant antibody, Abs-r-925. Complementarity determining regions of H-CDR1 through H-CDR3 are also shown in Table 9. It is further demonstrated in Example 3A of present invention.

Fig.5B

[16] [fig.5B] shows DNA sequence (SEQ ID NO:59) and amino acid sequence (SEQ ID NO:60) of Light Chain Variable Regions (L-CVR) from rabbit anti- human-PD-1 recombinant antibody, Abs-r-925. Complementarity determining regions of L-CDR1 through L-CDR3 are also shown in Table 10. It is further demonstrated in Example 3B of present invention.

Fig.5C

[17] [fig.SC] shows DNA sequence (SEQ ID NO:73) and amino acid sequence (SEQ ID NO:74) of Heavy Chain Variable Regions (H-CVR) from humanized anti-human-PD-1 recombinant antibody, Abs-h-925. Complementarity determining regions of H-CDR1 through H-CDR3 are also shown in Table 1 1 . It is further demonstrated in Example 3A of present invention.

Fig.5D

[18] [fig.SD] shows DNA sequence (SEQ ID NO:75) and amino acid sequence (SEQ ID NO:76) of Light Chain Variable Regions (L-CVR) from humanized anti-human-PD-1 recombinant antibody, Abs-h-925. Complementarity determining regions of L-CDR1 through L-CDR3 are also shown in Table 12. It is further demonstrated in Example 3B of present invention.

Fig.6A

[19] [fig.6A] shows DNA sequence (SEQ ID NO:61 ) and amino acid sequence (SEQ ID NO:62) of heavy chain variable regions (H-CVR) from rabbit anti- human-PD-1 recombinant antibody, Abs-r-926. Complementarity

determining regions of H-CDR1 through H-CDR3 are also shown in Table 13. It is further demonstrated in Example 3A of present invention.

Fig.6B

[20] [fig.6B] shows DNA sequence (SEQ ID NO:63) and amino acid sequence (SEQ ID NO:64) of Light Chain Variable Regions (L-CVR) from rabbit anti- human-PD-1 recombinant antibody, Abs-r-926. Complementarity

determining regions of L-CDR1 through L-CDR3 are also shown in Table 14. It is further demonstrated in Example 3B of present invention.

Fig.6C

[21 ] [fig.6C] shows DNA sequence (SEQ ID NO:77) and amino acid sequence (SEQ ID NO:78) of Heavy Chain Variable Regions (H-CVR) from humanized anti-human-PD-1 recombinant antibody, Abs-h-926. Complementarity determining regions of H-CDR1 through H-CDR3 are also shown in Table 15. It is further demonstrated in Example 3A of present invention.

Fig.6D

[22] [fig.6D] shows DNA sequence (SEQ ID NO:79) and amino acid sequence (SEQ ID NO:80) of Light Chain Variable Regions (L-CVR) from humanized anti-human-PD-1 recombinant antibody, Abs-h-926. Complementarity determining regions of L-CDR1 through L-CDR3 are also shown in Table 16. It is further demonstrated in Example 3B of present invention.

Fig.7

[23] [fig.7] shows summarizing data on competitive binding to PD-1 of four humanized anti-human-PD-1 recombinant antibodies, Abs-h-923, Abs-h- 924, Abs-h-925 and Abs-h-926 with Nivolumab in competition ELISA in Vitro. It is further demonstrated in Example 6 of present invention. Fig.8

[24] [fig.8] shows is a bar graph summarizing the results of a mixed lymphocyte reaction (MLR) assay performed on human PBMCs treated with anti-PD-1 antibodies Abs-h-923, Abs-h-924, Abs-h-925 and Abs-h-926. These results indicate that three of four humanized anti-PD-1 antibodies significantly induce increased levels of IL-2 compared to antibody isotype controls (Negative control), two PD-1 antibodies (reference controls) and Nivolumab (Positive control). It is further demonstrated in Example 7 of present invention.