WO2024138910A1

WO2024138910A1 - Anti-cd73 antibody and use thereof

Info

Publication number: WO2024138910A1
Application number: PCT/CN2023/083405
Authority: WO
Inventors: Bing Xia; Yang Wang; Yifan Yang; Yuhong Zhou; Ziping Wei
Original assignee: Bliss Biopharmaceutical Hangzhou Co Ltd
Current assignee: Bliss Biopharmaceutical Hangzhou Co Ltd
Priority date: 2022-12-26
Filing date: 2023-03-23
Publication date: 2024-07-04
Anticipated expiration: 2025-06-26
Also published as: CN120418292A

Abstract

Provided are an anti-CD73 antibody and use thereof. The anti-CD73 antibody is effectively capable for diagnosis and companion diagnosis of CD73 with high specificity and precision.

Description

Anti-CD73 Antibody and Use Thereof

Field of invention

The present invention relates to biological diagnosis. In particular, the present invention relates to the quantative detection of CD73 protein in pathological tissues and its application.

Prior arts

CD73 is Ecto-5'-nuc1eotidase (ecto-5'-NT) , a glycophosphatidylinositol-anchored receptor expressed by populations of immune cells, stromal cells, epithelial and endothelial cells, cancer cells, and exosomes. This enzyme catalyzes the conversion of adenosine monophosphate (AMP) to adenosine. CD73 is believed to play a role in maintaining endothelial integrity as well as in mediating the inhibitory function of regulatory B and T lymphocytes.

Elevated CD73 expression occurs frequently in human cancers and often correlates with a worse prognosis observed in tumors of lung, colorectal, breast, ovarian, renal and glioblastoma. Clinically, elevated CD73 levels in the tumor tissue of several cancer types, including breast, ovarian, and colorectal cancers (CRC) , are linked to poor patient survival, which underscores the crucial role of CD73 in tumor progression.

Therefore, the IHC diagnosis of CD73 has important guiding significance for targeted therapy of patients with tumors expressing CD73. However, the currently available commercial IHC antibodies against CD73 are usually not sensitive to detect CD73 in trace amounts and have strong background, which significantly reduces their capability for diagnosis and companion diagnosis. Furthermore, the commercial IHC antibodies usually do not have the sensitivity range and are not able to distinguish different CD73-expressing levels (CD73 high-medium-low) .

Content of the present invention

In the first aspect, the present disclosure provided an isolated antibody, or an antigen-binding portion thereof.

In some embodiments, the antibody comprises a heavy chain and a light chain, wherein, the heavy chain comprises a variable domain (VH) comprising a first heavy chain CDR (CDR-H1) , a second heavy chain CDR (CDR-H2) and a third heavy chain CDR (CDR-H3) comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain comprises a light chain variable domain (VK) comprising a first light chain CDR (CDR-K1) , a second light chain CDR (CDR-K2) and a third light chain CDR (CDR-K3) comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. The CDR sequences in the VH and VK domains herein are identified according to the Kabat numbering scheme.

In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence having at least 80%identity to the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence that is substantially homologous to the given amino acid sequence of SEQ ID NO: 7.

In some embodiments, the light chian variable domain comprises the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence having at least 80%identity to the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence that is substantially homologous to the given amino acid sequence of SEQ ID NO: 8.

In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 9, or the amino acid sequence having at least 80%identity to the amino acid sequence of SEQ ID NO: 9, or the amino acid sequence that is substantially homologous to the given amino acid sequence of SEQ ID NO: 9; the light chain comprises the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence having at least 80%identity to the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence that is substantially homologous to the given amino acid sequence of SEQ ID NO: 10.

In the second aspect, the present disclosure provided an IHC reagent comprising the isolated antibody or the antigen-binding portion thereof as described in the first aspect.

In the third aspect, the present disclosure provided a CD73 detection kit comprising the isolated antibody or the antigen-binding portion thereof as described in the first aspect, or the IHC reagent as described in the second aspect.

In some embodiments, the CD73 detection kit further comprises endogenous peroxidase, secondary antibody, chromogenic substrate and/or hematoxylin.

In some embodiments, the main component in each CD73 detection kit is a mouse anti-CD73 monoclonal antibody.

In some embodiments, the kit is intended for the qualitative detection of CD73 protein in formalin-fixed, paraffin-embedded (FFPE) tissues of solid tumors including but not limit to gastric cancer and non-small-cell lung cancer (NSCLC) .

In some embodiments, the kit is used for the Leica BOND-MAX fully automated IHC and ISH staining system as the primary antibody for IHC detection of FFPE samples. Following incubation with the anti-CD73 monoclonal antibody, the FFPE sample is incubated with linker antibody specific to the host species of the primary antibody and then is incubated with the secondary antibodies (several peroxidases conjugated complex capable of combining with linker antibody) . The enzymatic conversion of the subsequently added chromogen results in precipitation of a visible reaction product at the site of antigen. Then the sample is counterstained using hematoxylin, coverslipped, and interpreted using light microscopy.

In the fourth aspect, the present disclosure provided an isolated nucleic acid molecule encoding the isolated antibody or antigen-binding portion thereof as described in the first aspect.

In the fifth aspect, the present disclosure provided a recombinant expression vector comprising the isolated nucleic acid molecule as described in the fourth aspect.

In the sixth aspect, the present disclosure provided a transformant comprising an isolated nucleic acid molecule as described in the fourth aspect or a recombinant expression vector as described in the fifth aspect in a host cell.

In the seventh aspect, the present disclosure provided a method of manufacturing an antibody comprising a step of culturing the transformant as described in the sixth aspect; preferably, the method comprise the steps of (i) culturing the transformant as described in the sixth aspect under conditions suitable for the expression of the isolated antibody or antigen-binding portion thereof; and optionally (ii) isolating or obtaining the isolated antibody or antigen-binding portion thereof from the host cell or from the supernatant.

In the eighth aspect, the present disclosure provided the use of the isolated antibody or the antigen-binding portion thereof as described in the first aspect, in preparation of the IHC reagent as described in the second aspect.

In the ninth aspect, the present disclosure provided the use of the isolated antibody or the antigen- binding portion thereof as described in the first aspect, the IHC reagent as described in the second aspect, or the CD73 detection kit as described in the third aspect, for diagnosis and/or companion diagnosis of CD73.

In the tenth aspect, the present disclosure provided a method for diagnosis and/or companion diagnosis of CD73.

In some embodiments, the method comprises treating a prepared CD73 sample by the isolated antibody or the antigen-binding portion thereof as described in the first aspect, the IHC reagent as described in the second aspect, or the CD73 detection kit as described in the third aspect, under appropriate reaction conditions. Preferably, the CD73 sample is FFPE sample.

In some embodiments, the appropriate reaction conditions are as follow:

Brief description of the drawings

Fig. 1 shows CD73 detection result in sample Sto010 by antibody D7F9A.

Fig. 2 shows CD73 detection result in sample Sto010 by antibody A6F7.

Fig. 3 shows CD73 detection result in sample Sto015 by antibody D7F9A.

Fig. 4 shows CD73 detection result in sample Sto015 by antibody A6F7.

Detailed description of the preferred embodiment

In the present invention, the terms "antibody" and "immunoglobulin" , as used herein, refer broadly to any immunological binding agent that comprises an antigen binding domain, including polyclonal and monoclonal antibodies.

Thus, the term “antibody” includes immunological binding agents that comprise an antigen binding domain obtained from or derived from an antibody, e.g. obtained from or derived from an Ig (e.g. IgG) antibody.

In some embodiments, polyclonal antibodies are preferred (e.g. polyclonal antibodies that are generated in (or raised in or isolated from) an animal (e.g. a rabbit or mouse such as a specific pathogen free (SPF) rabbit or mouse) immunized with an isolated CD73 of the present invention.

In some embodiments, monoclonal antibodies are preferred (e.g. mouse monoclonal or human monoclonal antibodies or humanized monoclonal antibodies or rabbit monoclonal antibodies) .

Depending on the type of constant domain in the heavy chains, whole antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM and the antibodies of the invention may be in any one of these classes. Several of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. The heavy-chain constant domains that correspond to the difference classes of immunoglobulins are termed α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Generally, where whole antibodies rather than antigen binding regions are used in the invention, IgG (e.g. IgG2 or IgG4) and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.

The "light chains" of mammalian antibodies are assigned to one of two clearly distinct types: kappa (κ) and lambda (λ) , based on the amino acid sequences of their constant domains and some amino acids in the framework regions of their variable domains.

As will be understood by those in the art, the immunological binding reagents encompassed by the term "antibody" includes or extends to all antibodies and antigen binding fragments thereof, including whole antibodies, dimeric, trimeric and multimeric antibodies; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies, and fragments thereof.

The term "antibody" is thus used to refer to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F (ab') ₂, single domain antibodies (DABs) , TandAbs dimer, Fv, scFv (single chain Fv) , dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively) ; sc-diabody; kappa (lamda) bodies (scFv-CL fusions) ; BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells) ; DVD-Ig (dual variable domain antibody, bispecific format) ; SIP (small immunoprotein, a kind of minibody) ; SMIP ( "small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting" ) ; small antibody mimetics comprising one or more CDRs and the like.

The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. In some embodiments, the antibodies of the invention are non-human antibodies (e.g. rabbit or rat or mouse antibodies) . In some embodiments, the antibodies are mouse antibodies.

The term "heavy chain complementarity determining region" ( "heavy chain CDR" ) as used herein refers to regions of hypervariability within the heavy chain variable region (VH domain) of an antibody molecule. The heavy chain variable region has three CDRs termed heavy chain CDR1 (CDR-H1) , heavy chain CDR2 (CDR-H2) and heavy chain CDR3 (CDR-H3) from the amino terminus to carboxy terminus. The heavy chain variable region also has four framework regions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxy terminus) . These framework regions separate the CDRs.

The term "heavy chain variable region" (VH) as used herein refers to the variable region of a heavy chain of an antibody molecule.

The term "light chain complementarity determining region" ( "light chain CDR" ) as used herein refers to regions of hypervariability within the light chain variable region (VK domain) of an antibody molecule. Light chain variable regions have three CDRs termed light chain CDR1 (CDR-K1) , light chain CDR2 (CDR-K2) and light chain CDR3 (CDR-K3) from the amino terminus to the carboxy terminus. The light chain variable region also has four framework regions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxy terminus) . These framework regions separate the CDRs.

The term "light chain variable region" (VK) as used herein refers to the variable region of a light chain of an antibody molecule.

The CDRs of the antibodies of the invention are preferably separated by appropriate framework regions such as those found in naturally occurring antibodies and/or effective engineered antibodies. Thus, the VH, VK and individual CDR sequences are preferably provided within or incorporated into an appropriate framework or scaffold to enable antigen binding. Such framework sequences or regions may correspond to naturally occurring framework regions, FR1, FR2, FR3 and/or FR4, as appropriate to form an appropriate scaffold, or may correspond to consensus framework regions, for example identified by comparing various naturally occurring framework regions. Alternatively, non-antibody scaffolds or frameworks, e.g. T cell receptor frameworks can be used. Appropriate sequences that can be used for framework regions are well known and documented in the art and any of these may be used. In particular, framework regions that allow the maintenance of antigen specificity, for example framework regions that result in substantially the same or the same 3D structure of the antibody.

CDR sequences of antibodies of the invention may be CDR sequences in the VH domains and VK domains of antibodies of the invention as identified using any suitable method (or tool) , for example as identified according to the well-known methods of Kabat (e.g. Kabat, et al., "Sequences of Proteins of Immunological Interest" , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 647-669, 1991) or Chothia (e.g. Chothia C, et al. (1989) Nature, 342: 877–883, or Al-Lazikani et al., (1997) JMB 273, 927-948) .

In certain embodiments, the antibody or antibody fragment of the present invention comprises all or a portion of a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g. an IgG2 or an IgG4 heavy chain constant region, or a portion thereof. Furthermore, the antibody or antibody fragment can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region, or a portion thereof. All or part of such constant regions may be produced naturally or may be wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art. When a full complement of constant regions from the heavy and light chains are included in the antibodies of the invention, such antibodies are typically referred to herein as "full length" antibodies or "whole" antibodies. Thus, in some embodiments, the antibodies of the invention are Ig (e.g. IgG) antibodies.

Preferably, antibodies of the present invention (e.g. Ig antibodies) comprise two identical VH domains and two identical VK domains. Thus, in the case of Ig (e.g. IgG) antibodies for example, Ig antibodies typically comprise (or consist of) two identical heavy chains and two identical light chains, which together form the Ig antibody.

The antibodies or antibody fragments can be produced naturally or can be wholly or partially synthetically produced. Thus the antibody may be from any appropriate source, for example recombinant sources and/or produced in transgenic animals or transgenic plants, or in eggs using the IgY technology. Thus, the antibody molecules can be produced in vitro or in vivo.

Preferably, the antibody or antibody fragment comprises an antibody light chain variable region (VK) that comprises three CDR domains and an antibody heavy chain variable region (VH) that comprises three CDR domains. Said VK and VH generally form the antigen binding site.

Amino acid sequences that are “substantially homologous” to isolated peptides or proteins are sequences having, or sequences comprising, a sequence that has, 1, 2, or 3 amino acid substitutions or additions or deletions (preferably 1 or 2, more preferably 1) compared with the amino acid sequence of the given isolated peptide or proteins.

Amino acid sequences that are “substantially homologous” to isolated peptides or proteins include sequences having, or sequences comprising (or consisting of) a sequence that has, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%sequence identity to the given isolated peptide or protein sequence. Sequence identities of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%or at least 95%are preferred.

Alterations in the amino acid sequences can be with conservative or non-conservative amino acids. Preferably said alterations are conservative amino acid substitutions.

A "conservative amino acid substitution" , as used herein, is one in which the amino acid residue is substituted with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine) , acidic side chains (e.g. aspartic acid, glutamic acid) , uncharged polar side chains (e.g. asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine) .

The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid sequences of the present invention that perform substantially the same function as the proteins of the invention in substantially the same way. For example, any substantially homologous isolated peptide or protein should typically retain the ability to bind CD73 or a mutant form of CD73.

Methods of carrying out the above described manipulation of amino acids (e.g. to generate “substantially homologous” sequences) are well known to a person skilled in the art.

Homology (e.g. sequence identity) may be assessed by any convenient method. However, for determining the degree of homology (e.g. identity) between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22: 4673-4680, 1994) . Other methods that may be used to align sequences are the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48: 443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2: 482, 1981) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Other methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48: 1073, 1988) and those described in Computational Molecular Biology, Lesk, e. d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.

The term "nucleic acid sequence" or "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid molecules may be double stranded or single stranded. The nucleic acid molecules may be wholly or partially synthetic or recombinant.

The term "substantially homologous" as used herein in connection with a nucleic acid sequence includes sequences having at least 70%or 75%, preferably at least 80%or 85%, and even more preferably at least 90%, 95%, 96%, 97%, 98%or 99%, sequence identity to the starting nucleic acid sequence.

Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses) , so long as the vector is compatible with the host cell used. The expression vectors are "suitable for transformation of a host cell" , which means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner that allows expression of the nucleic acid.

The invention therefore contemplates an expression vector, e.g. a recombinant expression vector containing or comprising a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.

Expression vectors can be introduced into host cells to produce a transformed host cell. The terms "transformed with" , "transfected with" , "transformation" and "transfection" are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al., 1989 (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and other laboratory textbooks.

Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells. For example, the proteins of the invention may be expressed in yeast cells or mammalian cells. In addition, the proteins of the invention may be expressed in prokaryotic cells, such as Escherichia coli. The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis.

A yet further aspect provides an expression construct or expression vector or expression system (e.g. a viral or bacterial or other expression construct, vector or system) comprising one or more of the nucleic acid fragments or segments or molecules of the invention. Preferably the expression constructs or vectors or systems are recombinant. Preferably said constructs or vectors or systems further comprise the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention. Preferred constructs etc., are those which allow prolonged or sustained expression of the antibodies (or binding proteins) of the invention within the host target species, e.g. within pigs. Such expression can be transient, e.g. episomal, or more permanent, e.g. via genomic integration, providing sufficient levels and length of expression are achieved in order for a therapeutic or biological effect to be observed.

A yet further aspect provides a host cell (e.g. a mammalian or bacterial or yeast host cell) or virus comprising one or more expression constructs or expression vectors of the invention. Also provided are host cells or viruses comprising one or more of the nucleic acid molecules of the invention. A host cell (e.g. a mammalian host cell or bacterial host cell, or yeast host cell) or virus expressing an antibody (or binding protein) of the invention forms a yet further aspect.

Such expression constructs or vectors or systems, or host cells or viruses, or other nucleic acid products or fragments encoding the antibodies (or binding proteins) of the invention can be administered as therapeutic agents to a subject to allow the production of the antibodies (or binding proteins) of the invention in situ within the subject and thereby exert their therapeutic effects.

A yet further aspect of the invention provides a method of producing (or manufacturing) an antibody of the present invention comprising a step of culturing the host cells of the invention. Preferred methods comprise the steps of (i) culturing a host cell comprising one or more of the recombinant expression vectors or one or more of the nucleic acid sequences of the invention under conditions suitable for the expression of the encoded antibody or protein; and optionally (ii) isolating or obtaining the antibody or protein from the host cell or from the growth medium/supernatant. Such methods of production (or manufacture) may also comprise a step of purification of the antibody or protein product and/or formulating the antibody or product into a composition including at least one additional component, such as a pharmaceutically acceptable carrier or excipient.

In embodiments when the antibody or antigen-binding portion thereof of the invention is made up of more than one polypeptide chain (e.g. certain fragments such as Fab fragments or whole antibodies) , then all the polypeptides are preferably expressed in the host cell, either from the same or a different expression vector, so that the complete proteins, e.g. antibody proteins of the invention, can assemble in the host cell and be isolated or purified therefrom.

In another aspect, the invention provides a method of binding CD73, comprising contacting a composition comprising CD73 with an antibody or antigen-binding portion thereof of the invention. In yet another aspect, the invention provides a method of detecting CD73, comprising contacting a composition suspected of containing CD73 with an antibody of the invention, under conditions effective to allow the formation of CD73/antibody complexes and detecting the complexes so formed. In one sense, the two methods are equivalent to the method for diagnosis and/or companion diagnosis of CD73 comprising treating a prepared CD73 sample by the isolated antibody or the antigen-binding portion thereof of the present invention.

The following examples further illustrate the present invention, but the present invention is not limited thereto.

Below presents preferred embodiments of the present invention based on the drawings in order to illustrate the technical schemes of the present invention in detail.

Embodiment 1 Preliminary validation of CD73 antibodies

1. Selection of the CD73 antibodies and CD73 detection procedure

Three monoclonal antibodies in total were chosen based on the information available on the CD73 antigen and its expression patterns: two commercial monoclonal antibodies were ERP6114 (Cat. No.: ab133582, Abcam) and D7F9A (Cat. No.: 13160, Cell Signaling Technology) ; and one monoclonal antibody was A6F7, which was provided by Bliss Biopharmaceutical Co, Ltd. The amino acid sequences of full lengty of A6F7’s heavy chain and light chain are as shown in SEQ ID NOs: 9 and 10 respectively. The FFPE samples of gastric cancer and NSCLC were prepared for validation according to the SOP for IHC sampling.

Leica BOND-MAX (Cat. No.: E-GZ-A033, Leica Biosystems Newcastle Ltd. ) fully automated IHC and ISH staining system is used to detect the expression level of CD73 in the samples. The IHC Protocol F Shuwen used for staining procedure is as follow:

1) Incubation with Peroxide Block for 5 min;

2) Incubation with Primary Antibody for 20 min;

3) Incubation with MARKER for 15 min;

4) Incubation with Post Primary for 15 min;

5) Incubation with Polymer for 8 min;

6) Incubation with DAB for 0 min;

7) Incubation with DAB for 10 min;

8) Incubation with Hematoxylin for 5 min.

All above reagents, except Primary Antibody, are included in BOND^TM Polymer Refine Detection Kit (Cat. No.: DS9800, Leica Biosystems Newcastle Ltd. )

2. Validation of the ERP6114 and D7F9A

A working solution was prepared for both ERP6114 (dilution ratio: 1: 2000) and D7F9A (dilution ratio: 1: 200) according to their corresponding Instructions for Use (IFU) . 20 gastric cancer samples were used for validation according to the Standard Operation Procedure (SOP) for IHC detection.

Results: Under the same reaction condition, D7F9A showed a higher positive detection rate and better staining intensity compared with ERP6114. The D7F9A can detect different expression levels of CD73 antigen among the 20 gastric cancer samples.

3. Study on dilution series for EPR6114

Considering that the recommended dilution concentrations were different between ERP6114 and D7F9A, in order to confirm whether EPR6114 can reach the same detection rate and staining intensity as those of D7F9A, the dilution series for EPR6114 were adjusted to 1: 200, 1: 500, and 1: 1000. The gastric cancer samples in Part 2 of Example 1 were used for validation according to the SOP for IHC detection.

Results: The expression of CD73, when using ERP6114 under 1: 200 dilution, increased but was lower than D7F9A. Under 1: 500 and 1: 1000 dilution of ERP6114, the expression is even lower than that under 1: 200, suggesting that both the affinity and antibody titer for ERP6114 are inferior to D7F9A and thus the former is not a good working clone. Results obtained from the D7F9A clone were chosen as control for following validation.

4. Study on D7F9A in NSCLC

A working solution was made for D7F9A in 1: 200 dilution according to the IFU. The validation was done for 16 NSCLC samples according the SOP for IHC detection.

Results: D7F9A was able to detect different levels of expressions of CD73 for the NSCLC samples. The expression level of CD73 was established for the 16 NSCLC samples.

5. Study on dilution series for A6F7

The A6F7 was diluted in 1: 3000, 1: 5000, 1: 6000, and 1: 10000 for validation using the gastric cancer and NSCLC samples with different CD73 expression levels determined in Part 2 of Example 1 and Part 4 of Example 1.

Results: Similar staining performance was achieved for 1: 3000 and 1: 5000 while the former showed a stronger background and thus was not suitable for working solution. Staining intensity was weakened when using 1: 6000 and 1: 10000. Thus, the concentration of 1: 5000 dilution was chosen as the working solution for validation using the Leica BOND-MAX (Cat. No. E-GZ-A033) .

Embodiment 2 Validation for the production process and reaction

Every production process followed the SOP for the production process Ecto-5’-prime-nucleoticlase (CD73) Detection Kit (Immunohistochemistry Method) stated in the Good Manufacturing Practice (GMP) to ensure the quality of the product.

1. Validation for the reaction

According to the development procedure, the reaction of the kit was validated using the Leica BOND-MAX with the bond polymer refine detection system.

2. Study on antigen retrieval solution for the A6F7 antibody

Reaction condition: For ER1 (BOND Epitope Retrieval Solution 1, Leica, Cat. No.: ER1124803) and ER2 (BOND Epitope Retrieval Solution 2, Leica) , each was incubated at 100℃ for 20 min separately using the gastric cancer and NSCLC samples with different CD73 expression levels determined by Part 2 of Example 1 and Part 4 of Example 1. The staining procedure followed the IHC Protocol F Shuwen.

Results: The A6F7 showed a slightly stronger staining intensity for ER2 than ER1 under the same incubating condition. However, ER1 showed a cleaner background in the extracellular matrix and fibroblast which favors the interpretation. Thus, ER1 was chosen as the antigen retrieval solution for the A6F7 antibody.

3. Study on the incubation time with the primary antibody

The A6F7 antibody was incubated for 10 min and 20 min under the condition described in Part 2 of Example 2 using the gastric cancer and NSCLC samples with different CD73 expression levels determined in Part 2 of Example 1 and Part 4 of Example 1. The staining procedure followed the IHC Protocol F Shuwen.

Results: A higher expression of CD73 was found when incubating with A6F7 for 20 min compared to 10 min, thus we chose 20 min as the incubation time for the primary antibody.

4. Study on the blocking time for the endogenous peroxidase

Two time points, 5 min and 10 min were chosen as the blocking time. The incubation time for A6F7 was set at 20 min. The gastric cancer and NSCLC samples with different CD73 expression levels determined by Part 2 of Example 1 and Part 4 of Example 1 were used. The staining procedure followed the IHC Protocol F Shuwen.

Results: Similar staining intensity and background were seen for both 5 min and 10 min treatment. As the shorter time can speed up the detection, thus we chose 5 min as the blocking time for the endogenous peroxidase.

5. Study on the incubation period for secondary antibody

The incubation period for the secondary antibody was tested for 20 min and 30 min with 5 min of blocking time for the endogenous peroxidase. The gastric cancer and NSCLC samples with different CD73 expression levels determined by Part 2 of Example 1 and Part 4 of Example 1 were used. The staining procedure followed the IHC Protocol F Shuwen.

Results: An obvious stronger staining intensity was seen when incubating for 30 min, however, this also produced stronger background staining for extracellular matrix and fibroblast. Given a shorter incubation time can save time, thus we chose 20 min as the secondary incubation time.

6. Study on the incubation time with the chromogenic substrate

The incubation period for the chromogenic substrate was tested for 5 min and 10 min with 5 min blocking with the endogenous peroxidase, 20 min incubated with the A6F7 antibody and 20 min incubated with the secondary antibody.

Results: The 10 min incubation time showed a slightly stronger staining intensity compared to 5 min, however, 10 min incubation also produced a stronger background. Thus, 5 min was chosen as the incubation time with the chromogenic substrate.

7. Summary

According to the studies on the reaction conditions, the final reaction condition for the Ecto-5’-prime-nucleotidase (5’-NT) Detection Kit (Immunohistochemistry Method) is as follows:

Embodiment 3 Analytical Performance

According to the SOP for the production process Ecto-5’-prime-nucleoticlase (CD73) Detection Kit (Immunohistochemistry Method) , three batches of kits were manufactured with 5 kits per batch to study the analytical performance. Five analytical performance studies were done: immunoreactivity, sensitivity, specificity, precision, and stability.

1. Immunoreactivity

In normal tissue immunoreactivity study, a normal tissue combination chip (Cat. No.: MuFDA1021, Bioaitech) that contains 30 samples were used for testing their antigen reactions. In tumor tissue immunoreactivity study, a tumor tissue combination chip (Cat. No.: X090Mc01, Bioaitech) that contains 90 samples were used for testing their antigen reactions. According to the stainnig procedure determined by Example 2, the CD73 detection kit containing the antibody A6F7 was used for immunohistochemical staining.

Results: As shown in Table 1 and Table 2, CD73 was expressed in normal tissues and various tumor tissues to varying degrees, which was consistent with the expression level in literatures (See [1] The ectonucleotidases CD39 and CD73: novel checkpoint inhibitor targets. Immunol Rev. Author manuscript; available in PMC 2018 Mar 1; [2] Anti-CD73 in cancer immunotherapy: awakening new opportunities. Trends Cancer. Author manuscript; available in PMC 2017 Feb 1. Trends Cancer. 2016 Feb 1; 2 (2) : 95–109; [3] The Roles of CD73 in Cancer. Biomed Res Int. 2014; 2014: 460654; and [4] Prognostic significance and immune correlates of CD73 expression in renal cell carcinoma. J Immunother Cancer. 2020; 8 (2) : e001467) . Therefore, the detection kit has potent immunoreactivity.

Table 1 Results summary of normal tissue combination chip

Note: [1] vascular endothelial positive, [2] lymphocyte positive, [3] stroma positive, [4] fibrocyte positive.

Table 2 Results summary of tumor tissue combination chip

2. Specificity

106 gastric cancer tissue chip samples (FFPE samples, I-IV Stages) and excised samples collected outside the laboratory were used to evaluate the expression of CD73. According to the staining procedure determined by Example 2, the CD73 detection kit containing the antibody A6F7 was used for immunohistochemical staining.

Results: As shown in Table 3, the positive rate of CD73 in gastric cancer was 44.4%, which is consistent with 45.3%in the literature report (See Expression and clinical significance of CD73 and hypoxia-inducible factor-1α in gastric carcinoma. World J Gastroenterol. 2013 Mar 28; 19 (12) : 1912–1918) .

Table 3 Results of gastric cancer

3. Precision

Lung cancer and gastric cancer tissues with clear CD73 expression were used as test samples for continuous section (Lun057B, Lun058B, Sto010, and Sto015) . According to the staining procedure determined by Example 2, the CD73 detection kit containing the antibody A6F7 was used for immunohistochemical staining. Inter-batch testing was performed for three batches of kits. Out of the three batches, one batch was tested for inter-instrument, intra-batch, inter-operator, inter-day, intra-day, intra-run testing. Average negative agreement (ANA) , average positive agreement (APA) , overall agreement (OA) , and 95%CI were calculated using bootstrap method.

Results: As shown in Tables 4～7, CV%of the results were all less than 10%, therefore, the detection kit has good repeatability in inter-batch, intra-batch, inter-operator, inter-day, intra-day, testing.

Table 4 Resutls summary of inter-batch

Table 5 Results summary of intra-batch testing

Table 6 Results summary of inter-operator testing

Table 7 Results summary of inter-day and intra-day testing

4. Performance comparison with D7F9A

Lung cancer and gastric cancer tissues with clear CD73 expression were used as test samples for continuous section (Sto010 and Sto015) . According to the staining procedure determined by Example 2, both the D7F9A antibody and the CD73 detection kit containing the antibody A6F7 was used for immunohistochemical staining.

Results: as shown in Figs. 1-4, both in samples Sto010 and Sto015, antibody D7F9A has non-specific binding in the samples and therefore result in a strong background, compared to the antibody A6F7.

5. Summary

The abovementioned content summarized the Phase I product development process for the Ecto-5’-prime-nucleoticlase (CD73) Detection Kit (Immunohistochemistry Method) .

It is to be understood that the foregoing description of Examples is intended to be purely illustrative of the principles of the invention, rather than exhaustive thereof, and that changes and variations will be apparent to those skilled in the art, and that the present invention is not intended to be limited other than expressly set forth in the following claims.

Claims

An isolated antibody or antigen-binding portion thereof, comprising a heavy chain and a light chain, wherein,

the heavy chain comprises

a heavy chain variable domain (VH) comprising a first heavy chain CDR (CDR-H1) , a second heavy chain CDR (CDR-H2) and a third heavy chain CDR (CDR-H3) comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and

the light chain comprises

a light chain variable domain (VK) comprising a first light chain CDR (CDR-K1) , a second light chain CDR (CDR-K2) and a third light chain CDR (CDR-K3) comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.
The isolated antibody or antigen-binding portion thereof of claim 1, wherein, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence having at least 80%identity to the amino acid sequence of SEQ ID NO: 7.
The isolated antibody or antigen-binding portion thereof of claim 1, wherein, the light chian variable domain comprises the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence having at least 80%identity to the amino acid sequence of SEQ ID NO: 8.
The isolated antibody or antigen-binding portion thereof of any of claims 1-3, wherein, the heavy chain comprise the amino acid sequences of SEQ ID NO: 9, or the amino acid sequence having at least 80%identity to the amino acid sequence of SEQ ID NO: 9; the light chain comprises the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence having at least 80%identity to the amino acid sequence of SEQ ID NO: 10.
An IHC reagent comprising the isolated antibody or the antigen-binding portion thereof of any of claims 1-4.
A CD73 detection kit comprising the isolated antibody or the antigen-binding portion thereof of any of claims 1-4, or the IHC reagent of claim 5.
The CD73 detection kit of claim 6, further comprising endogenous peroxidase, secondary antibody, chromogenic substrate and/or hematoxylin.
An isolated nucleic acid molecule encoding the isolated antibody or antigen-binding portion thereof of claims 1-4.
A recombinant expression vector comprising the isolated nucleic acid molecule of claim 8.
A transformant comprising an isolated nucleic acid molecule of claim 8 or a recombinant expression vector of claim 9 in a host cell.
A method of manufacturing an antibody comprising a step of culturing the transformant of claim 10; preferably, the method comprise the steps of (i) culturing the transformant of claim 10 under conditions suitable for the expression of the isolated antibody or antigen-binding portion thereof; and optionally (ii) isolating or obtaining the isolated antibody or antigen-binding portion thereof from the host cell or from the supernatant.
The use of the isolated antibody or the antigen-binding portion thereof of any of claims 1-4 in preparation of the IHC reagent of claim 5.
The use of the isolated antibody or the antigen-binding portion thereof of any of claims 1-4, the IHC reagent of claim 5 or the CD73 detection kit of claim 6 or 7 for diagnosis and/or companion diagnosis of CD73.
A method for diagnosis and/or companion diagnosis of CD73 comprising treating a prepared CD73 sample by the isolated antibody or the antigen-binding portion thereof of any of claims 1-4, the IHC reagent of claim 5 or the CD73 detection kit of claim 6 or 7 under appropriate reaction conditions, preferably, the CD73 sample is FFPE sample.
The method of claim 14, wherein the appropriate reaction conditions are as below: