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Penumbra, abbreviated as “Pen” (the gene) or “pen” (the protein), was first cloned as a gene differentially expressed in erythroblasts by the present inventor in 1998 and further characterized and reported in 2000 (ref. 1), 2005 (ref. 2) and 2007 (ref. 3).
Pen is the newest member of the tetraspanin superfamily and is designated as tetraspanin 33, abbreviated as Tspan33, in GenBank. It is also a member of the recently recognized “eight-cysteine tetraspanin” subfamily (TspanC8)(ref. 4-6). It was envisaged at the time of the cloning of Pen that anti-pen monoclonal antibodies (mAbs) could be important tools in hematology and immunology since many widely used blood cell markers such as CD9, CD37, CD53, CD63, CD81, CD82 and CD151 were all tetraspanins while CD20 resembles tetraspanins in size and the overall domain organization (infra).
Tetraspanins are evolutionarily conserved transmembrane proteins (ref. 7-9). There are thirty-three tetraspanins in both mouse and human. All tetraspanins contain short intracellular amino- and carboxyl-termini, four transmembrane domains, two extracellular domains (ECD1 and ECD2) and conserved amino acids at key positions (ref. 10). The ECD2 of tetraspanin contains four, six or eight cysteines, allowing the formation of two, three or four disulfide bonds that play crucial roles in creating a special fold in the large ECD2. Some tetraspanins can self-associate laterally to form large “tetraspanin-enriched microdomains” (ref. 11-13). Several tetraspanins have been shown to interact with specific membrane proteins, usually via ECD2, and incorporate them into “tetraspanin-enriched microdomains” and thereby modulate the efficiency of signal transduction by specific membrane proteins (ref. 14).
The membrane protein partners of tetraspanins are diverse and include CD19 (associates with tetraspanin 28, a.k.a. CD81 and tetraspanin 27, a.k.a. CD82)(ref. 15-20), integrin α4β1 (associates with tetraspanin 28, tetraspanin 27, tetraspanin 30, a.k.a. CD63, and tetraspanin 25, a.k.a. CD53)(ref. 21), integrins α3β1 and α6β1 (associate with tetraspanin 24, a.k.a. CD151)(ref. 22), P-selectin (associates with tetraspanin 30, a.k.a. CD63)(ref. 23), and vascular cell adhesion molecule-1 or VCAM-1 (associates with tetraspanin 24)(ref. 24), to name just a few.
Other membrane proteins that also contain four transmembrane domains and two extracellular domains but not the conserved amino acids at key positions are not included in the tetraspanin superfamily. A notable example is the B cell marker CD20 (ref. 25), which is a member of the CD20/FcεRIβ superfamily. Although CD20 resembles tetraspanins in size (297 amino acids vs. 283 amino acids in pen) and domain organization (short intracellular amino- and carboxyl-termini, four transmembrane domains, one small ECD1 between the first and second transmembrane domains and one large ECD2 between the third and fourth transmembrane domains), it shares no conserved amino acids at key positions with tetraspanins and has only one disulfide bond in ECD2 (ref. 26, 27). Therefore, CD20 is not classified as a tetraspanin. It appears that the predecessors of the tetraspanin and the CD20/FcεRIβ superfamilies evolved separately soon after their debut. Of note, CD20 is the target of the highly effective therapeutic mAb, RITUXIMAB™, for non-Hodgkin's B cell lymphomas, chronic B cell leukemia and autoimmune disorders (ref. 27-29).
Very little is known about TspanC8 except that they all have eight cysteines in their ECD2 and that most TspanC8, including pen, can form a specific complex with the membrane protein “A Disintegrin And Metalloprotease 10” (ADAM10)(ref. 5, 6, 30). TspanC8 is required for the biosynthesis, maturation and trafficking of ADAM10 from endoplasmic reticulum to the Golgi apparatus and finally to the cell surface or other membrane compartments. In the absence of TspanC8, ADAM10 remains trapped in the endoplasmic reticulum after synthesis (ref. 5, 6, 30). This effect is quite dramatic visually (ref. 6). An interesting parallel is seen in CD19, another quasi-pan B cell marker beside CD20 and a component of B Cell Receptor or BCR complex (ref. 15-17, 20). Like ADAM10, CD19 also remains trapped in the endoplasmic reticulum in the absence of its companion, tetraspanin 28, a.k.a. CD81 (ref. 31).
The mouse and human pens are 97.2% identical in amino acid sequence and 98.3% identical in ECD2 (ref. 3). For comparison, the human β-globin and mouse β1-globin share only 80.3% identity in amino acid sequence and the human and mouse CD20 are only 73% identical in amino acid sequence. The near identity (97.2%) of mouse and human pens indicates that there is an exceptionally high evolutionary pressure against their sequence variation, more so than for β-globin and CD20.
A major obstacle in the study of pen and TspanC8 in general has been the lack of mAbs against these proteins, especially the native forms on living cells (ref. 5; p. 39763, line 41). The near identity of mouse and human pen amino acid sequences may be part to blame in the case of hpen, but the difficulty in recreating the native topology of TspanC8 in vitro sans cell membrane is probably the main reason. Although a few polyclonal antibody preparations became available briefly (e.g. Abcam, cat. no. ab87543; Santa Cruz, cat. no. sc-138518; Atlas Antibodies HPA020357), all were raised against synthetic peptides corresponding to the intracellular, carboxyl terminus or part of ECD2 of hpen and did not recognize the native hpen protein or the native ECD2 on living cells. There was also the issue of off-targets with polyclonal antibodies raised against synthetic peptides. Therefore, their utility was limited. Our own efforts at creating anti-Pen mAbs began well before 2013 and underwent many changes in protocols.
Due to the lack of antibodies with proven specificity for native pen, most studies so far have relied on Northern or Western analysis or quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)(ref. 3, 32). These studies revealed that Pen has a restricted expression pattern with most Pen mRNA found in bone marrow, spleen and kidney (ref. 3 and unpublished human data, present inventor). This contrasts with the ubiquitous or quasi-ubiquitous expression pattern of most other tetraspanins such as CD81 (ref. 8, 9). The detection of Pen mRNA and protein in bone marrow was initially thought to reflect their expression in erythroblasts (ref. 3). The expression of Pen mRNA in mouse spleen was also initially thought to be attributable to erythroblasts as there is significant extramedullary erythropoiesis in the spleens of adult mice. However, the level of PEN mRNA in human spleen is also very high despite the fact that there is no extramedullary erythropoiesis in normal adult human spleens (ref. 3). This apparent contradiction indicates that an additional cell type(s) in the spleen may express mouse Pen or human PEN mRNA. In this regard, recent DNA array data, RT-PCR and Western blot analyses indicated that in vitro activated (by anti-CD40 mAb plus IL-4, or CD40 ligand plus IL-4, or CpG plus pokeweed mitogen/PWM plus pansorbin) mature yet naive human B cells (i.e. fully differentiated B cells with functional B Cell Receptor but have not encountered the corresponding antigens yet) upregulated human PEN mRNA compared with resting B cells (ref. 32). A similar pattern of mouse Pen expression was noted in resting and in vitro activated (by lipopolysaccharide/LPS plus IL-4) mouse D cells (ibici).
In this application, we describe the establishment of mAbs as exemplified by 29A6.2, 41B10.13 and 59E6.8 and their chimeric mouse-human IgG1 versions mh29A6.2, mh41B10.13 and mh59E6.8, which secret mAbs recognizing both mouse and human pen proteins (abbreviated as mpen and hpen according to the protein naming convention) in living cells specifically. Using these mAbs we are able to demonstrate for the first time that pen is expressed on the surface of virtually all CD19+ or CD20+ or B220+ B cells regardless of their activation status in all primary and secondarily lymphoid tissues examined as well as in peripheral blood. However, its expression profile within the B lymphocyte population is very different from that of CD19 or CD20 in that it has a very wide range of expression level. In addition, these mAbs revealed that pen is also expressed in a very small subpopulation of erythrocytes and/or erythroblasts in bone marrow and a subset of newly released erythrocytes in peripheral blood, consistent with the previous report (ref. 3). Furthermore, anti-pen mAbs dramatically reduced the number and size of lymphoid follicles in the spleen and shrank the size of the spleen by about 50% when they were administered in vivo for 5-7 days. Thus these anti-pen mAbs enabled us to establish pen as a new cell surface marker of human and mouse B lymphocytes and provide new tools for research, diagnosis, prognostication and therapy in immunology, hematology and oncology.
This Invention relates to the creation of anti-penumbra (tetraspanin 33; mouse and human) mAbs and the surprise findings that they recognize virtually all human and mouse B lymphocytes and that pen has a wider range of expression level on B lymphocytes than other B cell markers such as CD19 and CD20.
In one embodiment, the invention provides an IgM mAb “29A6.2” that binds to both human and mouse pen protein on living cells: a heavy chain with the complete variable region amino acid sequence (including signal peptide) corresponding to SEQ ID NO:1 and a kappa light chain with the complete variable region amino acid sequence (including signal peptide) corresponding to SEQ ID NO:4.
In another embodiment, the invention provides an IgM mAb “41B10.13” that binds to both human and mouse pen protein on living cells: a heavy chain with the complete variable region amino acid sequence (including signal peptide) corresponding to SEQ ID NO:2 and a kappa light chain with the complete variable region amino acid sequence (including signal peptide) corresponding to SEQ ID NO:4.
In an additional embodiment, the invention provides an IgM mAb “59E6.8” that binds to both human and mouse pen protein on living cells: a heavy chain with the complete variable region amino acid sequence (including signal peptide) corresponding to SEQ ID NO:3 and a kappa light chain with the complete variable region amino acid sequence (including signal peptide) corresponding to SEQ ID NO:4.
In still another embodiment, the invention provides chimeric mouse-human IgG1 mAbs “mh29A6.2”, “mh41B10.13”, and “mh59E6.8”, in which the variable regions of heavy and light chains of mAbs 29A6.2, 41B10.13 and 59E6.8 have been grafted in frame onto the constant domains of human IgG1 heavy chain and kappa light chain. These chimeric antibodies retain the pen-binding specificity of mouse mAbs but adopt the structures, properties and functions of the constant regions of human IgG1 and kappa light chain and greatly increase their potential as diagnostic tools and immunotherapy drugs in humans.
In the description that follows, a number of terms and conventions commonly used in immunology and molecular biology are utilized. In order to provide a clear and precise understanding of the specifications and claims, including the scope to be given such terms, the following definitions are provided.
Following the convention of protein and gene naming and used herein, “pen” refers to penumbra protein of any species. “mpen” refers to mouse penumbra protein specifically. “hpen” refers to human penumbra protein specifically. Pen is the abbreviation of the mouse Penumbra gene while PEN refers to human PENUMBRA gene. Tetraspanin 33 is the alternative name for penumbra in GenBank, abbreviated as Tspan33. As used herein, “protein”, “peptide”, “peptide fragment” and “partial fragment of protein or antibody” include polymers of three or more amino acids linked by consecutive peptide bonds. No distinction based on length is intended between a protein, a peptide, a peptide fragment or a partial fragment of protein (including antibody). The term “antibody” is art-recognized terminology and includes intact or active partial fragments of antibodies that bind to target antigens. The term immunoglobulin (Ig) may be used in place of antibody. Each heavy chain and each light chain of a naturally occurring antibody contains one “variable region” (V region or VR) located in the amino terminal portion of the peptide chain (VH or VL, for heavy or light chain variable regions, respectively). Each VR is about 110 amino acids long (excluding the N-terminal signal peptide) and contains regions of variability in amino acid sequences that distinguish the antibody made by one B (or plasma) cell clone from that of a different clone. The VR of one heavy chain is juxtaposed with the VR of one light chain for form an antigen-binding site. Most of the amino acid sequence differences among different antibodies are localized to three short segments in the VRs of heavy and light chains and termed the “hypervariable region”. Hypervariable regions are about ten amino acids long each, and they are held in place by more conserved amino acid sequences known as the “framework regions” (FR) of the VR. In each pair of disulfide-bonded heavy and light chains, the three hypervariable regions of a VL domain and the three hypervariable regions of a VH domain cluster together to form a three-dimensional antigen-binding surface. The hypervariable regions are also called “complementarity-determining regions” (CDRs) as they together create a three-dimensional surface that is complementary to the topology of the cognate antigen or epitope like a lock and its key. Fab stands for “fragment, antigen binding” and consists of a complete light chain coupled to a VH and the first constant region of the heavy chain. Therefore, Fab retains the ability to bind to its cognate antigen. Fabs that retain the heavy-chain hinge are called Fab′; when the interchain disulfide bonds are preserved, the two Fab′ fragments remain linked to produce a divalent form called F(ab′)2. Both Fab′ and F(ab′)2 can recognize its cognate antigen(s). In this application, the term “antibody” is used in its broadest sense and specifically covers, but is not limited to, monoclonal antibodies, polyclonal antibodies, multi-specific antibodies as well as their active partial fragments. Examples of active partial fragments of antibodies that bind to target antigens include, but not limited to, Fab, F(ab′)2, “domain antibody fragment” (VR alone of a heavy or light chain) and “diabody” (ref. 33). The term “monoclonal antibody” (mAb) as used in the art refers to a preparation of homogeneous antibody molecules and is used to differentiate it from “polyclonal antibody”. A chimeric antibody refers to any antibody or partial antibody fragments composed of genetically engineered amino acid sequences derived from two or more antibodies from different animal species (or strains) or different classes or subclasses of Igs, so long as they exhibits the desired biological activities. A “humanized antibody” refers to an antibody created by transferring the immunoglobulin variable or hypervariable region (plus possibly some FR) amino acid sequences or corresponding cDNAs or genes of a nonhuman antibody into the corresponding regions of human immunoglobulin amino acid sequences or cDNAs or genes. Almost all murine mAbs can be humanized by grafting their CDRs onto the FR of human antibodies. A humanized antibody has the advantage of being less likely to trigger adverse immune responses when given to humans and will work more effectively with the immune system of the recipients. “Conservative amino acid substitution” refers to substitution of amino acids that, as known to those skilled in the art, may be made generally without altering the biological activity of the resultant molecule. Examples of conservative amino acid substitution include, but not limited to, substitution of Ala with Gly or Ser; Arg with Lys or His; Asn with Gln or His; Asp with Glu or Asn; Cys with Ser or Ala; Gln with Asn; Glu with Asp or Gln; Gly with Ala; His with Asn or Gln; Ile with Leu or Val; Leu with Ile or Val; Lys with Arg or His; Met with Leu or Ile or Tyr; Phe with Tyr or Met or Leu; Pro with Ala; Ser with Thr; Thr with Ser; Trp with Tyr or Phe; Tyr with Trp or Phe; Val with Ile or Leu. Antibodies can be covalently (cleavable or noncleavable in vivo) linked to drugs or other functional groups (e.g. drugs, ligands, receptors, binding proteins, enzymes, fluorochromes, radioisotopes, metal particles) to serve various purposes while exploiting the antigen-antibody specificity. They can also be modified covalently (e.g. PEGylation, glycosylation) or noncovalently (e.g. ionic interaction) to alter their behavior in vivo such as stability, volume of distribution, renal ultrafiltration, lipid solubility, blood-brain barrier crossing, metabolism and antigen processing by particular cell types or enzymes.
As used herein, “erythrocytes” refers to “red blood cells” (already enucleated) and “erythroblasts” refers to erythroid precursors that still contain nuclei and have not completed the entire terminal differentiation process. “Young erythrocytes” refers to new erythrocytes in blood circulation or spleen and have just been released from bone marrow microenvironment. They are similar the so-called “shift cells”. “MNCs” stands for “mononuclear cells”, which are obtained by density-gradient centrifugation fractionation of blood, marrow or spleen cell preparations. “ACK lysis” refers to removal of erythrocytes by lysis using a hypotonic buffer containing ammonium chloride and potassium bicarbonate.
The following MATERIALS AND METHODS were used in the examples that follow.
Establishment of cell lines stably expressing cell surface mpen or hpen.
The cloning of the cDNA of mouse Pen (mPen) and human PEN (hPEN) has been described by the present inventor (ref. 3). The construction of the pcDNA3.1/mpen vector has also been described (ref. 3). Briefly, the entire coding region of mpen was cloned into the HindIII-XhoI sites of the pcDNA3.1 expression vector (Invitrogen). The resultant vector expressed mpen as a fusion protein with a Myc-His×6 tag in the intracellular C-terminus. The vector was electroporated into the pen-negative mouse “pre-B” cell line BaF3 (American Type Culture Collection or ATCC; BaF3 does not express mpen) and transfected cells were selected with G418 (1 mg/ml) for 10 days as described (ref. 3). Stable transtectants, hitherto referred to as “BaF3/mpen”, was first established in 2004 and used in the current anti-pen mAb project. Stable BaF3/mpen were screened for expression of the fusion protein by immunofluorescence staining of methanol-fixed cytospin preparations using purified anti-Myc mAb conjugated with rhodamine (Santa Cruz Biotechnology), followed by fluorescent microscopy. Immunoprecipitation using an anti-Myc mAb (clone 9E10)(Santa Cruz Biotechnology) and anti-mpen C-terminus rabbit antisera demonstrated that the stable transfectants produced the 39-kilodalton full-length mpen fused to a Myc-Hisx6 tag as predicted. As a negative control, BaF3 was transfected with the negative control vector pcDNA3.1 and selected with G418 (1 mg/ml) for 10 days to produce a stable line BaF3/pcDNA3.1. An hpen expression vector was constructed by ligating the entire coding sequence of hpen into the BgIII-HpaI sites of the retroviral expression vector MSCV-PGK-EGFP (ref. 34). The hpen protein was expressed from the long-terminal repeat (LTR)-enhancer of MSCV. The enhanced green fluorescent protein (EGFP) was expressed independently from the internal phosphoglycerate kinase (PGK) promoter. BaF3 was transfected with MSCV-hpen-PGK-EGFP (or the parental vector MSCV-PGK-EGFP as a negative control) and pSV2Neo (as a selectable marker) at 10:1 molar ratio using GenePulser Xcell (BioRad). Transfected cells were selected with G418 (1 mg/ml) for 10 days to obtain stable transfectants, hitherto referred to as BaF3/hpen. BaF3 and its derivatives were cultured in RPMI medium (Gibco) supplemented with fetal bovine serum (FBS; 10% vol/vol)(Hyclone), 2-mercaptoethanol (2-ME; 1×10−6 M)(Sigma) and WEHI 3B conditioned medium (10% vol/vol) as a source of mouse interleukin-3 (IL-3).
Four- to six-weeks-old female C57Black/6 (“B6”) or Balb/C or Pen “knockout” (in-frame deletion of first and second TMs; ref. 3) mice were immunized intraperitoneally with 1×107 BaF3/mpen (or BaF3/hpen) emulsified in complete Freund's adjuvant (CFA; MP Biomedicals) and boosted with 2-4×106 BaF3/mpen (or BaF3/hpen) intravenously without adjuvant. Four days after the boost, spleens were harvested and single cell suspension was prepared by lysis with ammonium chloride lysis buffer (ACK, 017.2) and fused with SP2/0 AG14 myeloma cells (ATCC) using polyethylene glycol (Roche), plated in 96-well plates and selected with HAT (hypoxanthine, aminopterin and thymidine)(Sigma). Supernatants of hybridomas were screened by indirect immunofluorescence using BaF3/mpen as positive indicators and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulins (GAMIgs)(BD Pharmingen) as the secondary antibody. Positive supernatants were rescreened with BaF3 and BaF3/pcDNA3.1 to exclude non-pen-specific clones. Pen-specific clones were subcloned by limiting dilution at 0.5 cells/well, rescreened and subcloned again by limiting dilution at 0.2 cells/well. Wells were inspected after seeding to identify those with only one cell. Clonal lines were maintained in RPMI supplemented with 10% FBS, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 0.01 M, pH 7.2)(Fisher Biotech) and 2-ME (1×10−5 M). The class of antibody was determined using the ISOSTRIP™ mouse monoclonal antibody isotyping kit (Roche Diagnostics)
Purification and Conjugation of mAbs.
mAbs were purified from tissue culture supernatant using agarose-recombinant protein A (Abcam), which does not bind bovine serum albumin or bovine Igs but has variable binding capacity for mouse IgM. Purified antibodies were conjugated with FITC per manufacturer's instructions (Abcam).
Preparation of Hematopoietic Cells from Spleen, Bone Marrow, Lymph Node and Blood.
Spleen, bone marrow, lymph node and peripheral blood cells were obtained from Balb/C or B6 mice and treated with ACK lysis buffer to remove most erythrocytes. Alternatively, erythrocytes (as well as polymorphonuclear cells) were removed by density gradient centrifugation through a step-gradient of NYCOPREP™ (p=1.077 g/ml)(Nycomed). In some experiments, whole bone marrow was analyzed without ACK lysis or NYCOPREP™ density centrifugation. For human hematopoietic cells, heparinized blood was diluted with RPMI at a 1:5 ratio and centrifuged through a step-gradient of FICOLL-PAQUE™ Plus (p=1.077 g/ml)(Amersham Pharmacia Biotech). Light-density cells (ρ<1.077 g/ml) were collected and washed before staining with antibodies or culturing.
The medium used for cell staining was Hank's Balanced Salt Solution (HBSS) supplemented with heat-inactivated FBS (5% vol/vol) and sodium azide (0.09% wt/vol). This solution is known as “H5FAH”. All staining and washing were performed at 4° C. The flow cytometer used was a DxP flow cytometer with CYTOTEK™ upgrade (Cytotek). Analysis of flow cytometry data was performed using FLOWJO™ v. 9 (
Four to six million light-density (ρ<1.077 g/ml) human peripheral blood mononuclear cells (MNC) were cultured in 2 ml of RPMI supplemented with 20% FBS, glutamine, nonessential amino acids, sodium pyruvate, HEPES and 2-ME (1×10−6 M), human IL4 (hIL-4; 20 ng/ml)(R&D Systems) without or with human IL-2 (hIL-2; 2 ng/ml)(Chiron) plus or minus human CD40 ligand (hCD40L; 200 ng/ml)(BioLegend) or PWM (20 μg/ml)(Sigma). Human B cells do not express Toll-Like Receptor 4 (TLR4) for LPS but can be activated by PWM. Mouse spleen MNCs were cultured in the same medium supplemented with hIL-4 (20 ng/ml) without or with hIL-2 (2 ng/ml) and PWM or E. coli LPS (20 ng/ml)(Sigma). Cells were analyzed after culturing for 24-72 hrs.
All anti-pen hybridomas described in this application resulted from the fusion of the spleen cells of B6 mice with the myeloma cell line SP2/0 AG14 (Balb/C origin). Therefore F1 progenies from Balb/C×B6 crosses were used as the hosts in implantation studies to prevent rejection of hybridomas. To minimize variables, only female littermates were used in each study. Five to 10 days before implantation of hybridomas, each mouse was given an intraperitoneal injection of 0.1 ml of Incomplete Freund's Adjuvant (IFA)(MP Biomedicals) emulsified with phosphate buffered saline (PBS; pH7.2) to induce ascites to support hybridoma growth. Mice were then injected with 5×106 hybridoma cells (either 29A6.2 or the parental line SP2/0 AG14) intraperitoneally. After 5-10 days mice were euthanized and spleens and kidneys were photographed and weighed. The kidneys and spleens were fixed in 3.7% formaldehyde in PBS and embedded in paraffin. Paraffin sections were stained with hematoxylin-eosin (HE stain)(Sigma).
Determination of the cDNA and Amino Acid Sequences of the Entire Heavy- and Light-Chain Variable Regions of Anti-Pen Hybridomas.
cDNA syntheses and rapid amplification of cDNA ends were performed using poly(A)+ mRNAs and primers located in the constant regions of mouse IgM heavy- and kappa light-chain constant region 1. The 5′ ends of cDNAs were dC-tailed using terminal deoxynucleotidyl transferase (BioLab). 5′ rapid amplification of cDNA ends (5′ RACE) was performed using nested constant region 1 (of p heavy chain or K light chain)-specific primers and 5′ RACE Abridged Anchor Primer (Life Technologies) that incorporated desired restriction sites in the 5′ or 3′ ends. PCR products were digested with appropriate restriction enzymes and cloned into the EcoRI/SalI (for light chains) or BamHI/SalI (for heavy chains) sites of pBluescript and plasmid DNAs (five or more clones per heavy- or light-chain) were prepared and Sanger sequencing was performed using T3 and T7 primers. Sequences were checked against all known databases using the BLAST program to verify their uniqueness and translatability. Signal peptides were identified using the SignalP-4.1 program (cbs.dtu.dk/services/SignalIP-4.1).
Creation of Chimeric Mouse-Human IgG1 Anti-Pen mAbs.
The entire coding sequences (including start codons and signal peptides) of the variable regions of the heavy- and light-chain cDNAs of hybridoma 29A6.2, 41B10.13 and 59E6.8 were amplified by PCR using Elongase (Invitrogen) from corresponding, full-length 5′ RACE cDNA clones in Bluescript and ligated in frame into the EcoRI-NheI sites of pFUSE-CHIg-hG1 vector (InvivoGen) to create chimeric heavy-chains consisting of mouse heavy-chain variable regions fused to the human IgG1 heavy-chain constant region, and into the BstEII/BsiWI sites of pFUSE2-CLIg-hk (InvivoGen) to create chimeric light-chains consisting of mouse light-chain variable regions fused to human kappa light-chain constant region. A Kozak consensus sequence (5′GCCGCCACC) was inserted immediately upstream of the start codon to Improve protein translation. The chimeric constructs were Sanger sequenced to ensure correct coding. Chimeric mouse-human heavy- and light-chain expression constructs were co-transfected into the HEK293 cell line and selected with Zeocin and/or Blasticidin and/or G418 (if pSV2Neo is co-transfected). Multiple clonal lines were established for each pair of chimeric heavy and light chains. The chimeric mouse-human mAbs were named according to the origin of the heavy and light chains with a prefix mh. Thus “mh29A6.2” refers to antibodies produced by HEK293 expressing the heavy- and light-chain variable regions of hybridoma 29A6.2 fused with human IgG1 heavy-chain and kappa light-chain constant domains, respectively. The entire antigen-binding site of 29A6.2 is reconstituted in “mh29A6.2” but the rest of antibody is derived from human IgG1/kappa light chain. To test the antigen-binding activity of chimeric antibodies, supernatants of individual HEK293 transfectants (clones) were incubated with BaF3/mpen or hpen that were pretreated with FcBlock. After washing, bound chimeric antibodies were detected by a FITC-conjugated mAb against human IgG Fc domain (clone HP6017; BioLegend). The supernatants of empty (control) vector-transfected HEK293 were used as negative controls.
Spleen cells of immunized mice were fused with the myeloma cell line SP2/0 AG14 and selected with HAT for 10-14 days. Supernatants from all wells with hybridomas were screened by indirect immunofluorescence using BaF3/mpen as the positive indicator and BaF3 and BaF3/pcDNA3.1 as negative controls. Multiple clones fulfilling such criteria were subcloned by limiting dilution, expanded and further characterized. Three independent clones, 29A6.2, 41B10.13 and 59E6.8, are described in this application, although many more positive wells were identified in the primary screen. As shown in
mAb 29A6.2 (or 41B10.13 or 59E6.8) recognized cell surface mpen in BaF3/mpen in immunofluorescence microscopy (
mAbs 29A6.2, 41B10.13 and 59E6.8 were purified from tissue culture supernatant using agarose-recombinant protein A and conjugated with FITC. Mouse spleen cells were treated with ACK lysis buffer to remove RBC, pre-incubated with FcBlock and stained with FITC-conjugated mAb plus a PE-conjugated lineage-specific antibody and analyzed by flow cytometry. As shown in
In the initial study, all live cells (based on exclusion of the fluorescent dye 4′,6-diamidino-2-phenylindole or DAPI) were included in the flow cytometry analyses to establish the complete spectrum of hematopoietic cells that expressed cell surface pen. The analyses were repeated on live “lymphocyte”-gated cells only (as defined by forward and side scatters; also contains non-lymphocytes). The findings were essentially the same or very similar. Therefore, most analyses focused on live “lymphocyte”-gated populations unless stated otherwise.
Anti-Pen mAbs Recognize B Lymphocytes Regardless of their Activation Status.
To study the effect of B lymphocyte activation on the expression of pen, mouse spleen MNCs were stimulated in vitro with IL-4 and E. coli LPS (with or without IL-2). Cells were harvested 24-72 hrs. after stimulation and stained with 29A6.2-FITC (or 41B10.13-FITC or 59E6.8) and CD19-PE and analyzed by flow cytometry. As shown in
The results of flow cytometry analyses of mouse bone marrow cells (after lysis of most erythrocytes by ACK buffer) were similar to those of spleen cells except that a higher fraction of CD19+ B cells expressed lower levels of pen (
Mouse blood cells were lysed by ACK buffer and treated with FcBlock and stained with various antibodies. As shown in
Single cell preparations of omental and cervical lymph nodes were subjected to ACK buffer lysis, treated with FcBlock and stained with FITC-conjugated anti-pen mAbs and PE-conjugated lineage-specific antibodies. The results again showed that most of the CD19+ or CD20+ lymph node B lymphocytes also expressed pen (
mAbs 29A6.2, 41B10.13 and 59E6.8 Recognize Human CD19+ B Cells in Blood.
As illustrated in
mAbs 29A6.2, 41B10.13 and 59E6.8 Recognize Human B Cells Regardless of their Activation Status.
Unlike mouse B cells, human B cells do not express the toll-like receptor 4 (TLR4; the receptor for LPS) and therefore cannot be activated by LPS. However, human B cells can be activated in vitro by hCD40L plus hIL-4 or PWM plus hIL-4. Therefore we compared the staining of human B cells with anti-hCD19-PE plus 41B10-FITC before and after stimulation with hCD40L plus hIL-4 for 3 days. As shown in
The ability of anti-mpen mAbs to recognize both human and mouse cell-surface pen and the conserved expression patterns in both species made it possible to extrapolate or predict the biological effects of anti-pen mAbs in humans by examining their effects in mice. To study the possible effects of mAb 29A6.2 on mouse splenic B cells, we implanted the hybridoma 29A6.2 cells intraperitoneally in F1 progenies of B6×Balb/C crosses. As hybridoma 29A6.2 shares the genetic background of Balb/C (SP2/0 AG14) and B6 (fusion partner) mice, the hybridoma cells or their secreted immunoglobulins would not be rejected by the B6×Balb/C F1 progenies. To support the growth of the implanted hybridomas, the recipient F1 mice were injected with incomplete Freund's adjuvant (IFA) to induce ascites 5 days prior to implantation of hybridomas or SP2/0 AG14 (negative control). After 7-10 days, spleens were harvested, weighed, photographed, fixed in formaldehyde and the sections examined microscopically.
As shown in
Determination of the Nucleotide and Amino Acid Sequences of the Entire Variable Regions of mAbs 29A6.2, 41B10.3 and 59E6.8.
Complementary DNAs (cDNAs) of the entire VRs of heavy and light chains were obtained by 5′ RACE and cloned into pBluescript and Sanger sequenced. These cDNAs contain the 5′ untranslated regions, the entire coding sequences of the VRs including the start codons, the signal peptides and the first parts of the constant regions. The complete amino acid sequences of the VRs (including signal peptides) of the heavy- and light-chains of mAbs 29A6.2, 41B10.13 and 59E6.8 were deduced from the cDNA sequences. The signal peptides of all chains were identified using SignalP-4.1 software.
SEQ ID NO:1-3 correspond to the entire VR amino acids sequences (including signal peptides) of the p heavy chains of mAbs 29A6.2 (SEQ ID NO:1), 41B10.13 (SEQ ID NO:2) and 59E6.8 (SEQ ID NO:3). In all three, the first 19 amino acids represent the signal peptides that are removed by signal peptidase in the mature p heavy chains.
SEQ ID NO:4 corresponds to the entire VR amino acid sequences (including signal peptide) of the K light chains of mAbs 29A6.2, 41B10.13 and 59E6.8. These three mAbs share the same K light chain but their heavy chains are different. Amino acids 1-19 of SEQ ID NO:4 represent the signal peptide of the K light chain that is removed by signal peptidase in the mature K light chain.
Creation of Chimeric Mouse-Human IgG1 Chimeric mAbs by Recombinant DNA Technology.
Chimeric mouse-human IgG1 mAbs were constructed as detailed in MATERIALS AND METHODS.
This invention describes the creation and characterization of anti-mpen mAbs and the demonstration that anti-mpen mAbs described in this application recognize human pen (hpen) equally well. Furthermore, these mAbs recognize the native pen protein displayed on living human and mouse cells. Importantly, these mAbs recognize virtually all human and mouse CD19+ or CD20+ or B220+ B lymphocytes and do so regardless of their activation status. In addition, we have determined the nucleotide and amino acid sequences of the entire VRs of the heavy and light chains of these mAbs including their signal peptides. Finally, using recombinant DNA technology, we created chimeric mouse-human IgG1 mAbs consisting of the entire VRs of the mouse mAbs grafted in frame onto the constant regions of human IgG1 heavy chain and kappa light chain.
The creation of anti-pen mAbs followed many years of research, starting with the cloning of the human and mouse Penumbra genes (ref. 1-3), followed by the elucidation of their domain structures (ref. 3) and more recently, the identification of their membrane protein partners (ADAM-10) and their possible role in the proteolytic activation of the Notch receptor (upon binding of Notch ligands)(ref. 4-6) and possibly other signaling events. These early efforts provided important clues as to how best to immunize and screen for antibodies that could recognize native pen on living cells. However, the cloning of the human and mouse Penumbra genes was the crucial event.
Although anti-pen mAbs recognize virtually all CD19+ or CD20+ B lymphocytes, they exhibit a very different staining profile than anti-CD19 or anti-CD20 mAbs. While staining with fluorochrome-conjugated anti-CD19 mAbs resulted in relatively uniform fluorescence intensity in the CD19+ population (usually within one log or a 10-fold range)(
Another advantage of the anti-pen mAbs described in this disclosure is that since the same mAbs can recognize both mouse and human pens, it is possible to predict some of the behavior and side effects of these mAbs (or the chimeric human-mouse versions) in humans by studying the existing mAbs in mice. This may save a lot of time, effort and expense in preclinical studies of anti-pen mAbs.
A significant advantage of the chimeric mouse-human IgG1 anti-pen mAbs described in this application is that that the human IgG1 Fc portion can bind to protein A or G or a number of IgG1 Fc-binding proteins more efficiently. This will make facilitate the purification of these chimeric mAbs. Another advantage is that the human IgG1 Fc does not bind to mouse FcγIIIR. Thus, the chimeric mouse-human mAbs described in this application may be used to study mouse blood cells without pretreatment of mouse cells with FcBlock. The chimeric antibodies also have advantages over IgM mAbs in applications that benefit from the smaller size of the chimeric IgG1 antibodies. Importantly, human IgG1 has a stronger potential for antibody-dependent cell-mediated cytotoxicity (ADCC) in humans, which translates into more effective killing of target cells. Finally, the 293 HEK cells are of human origin. Therefore, the chimeric mAbs purified from these producer cells are less likely to contain impurities that may trigger adverse immune reactions when administered into humans.
The anti-pen mAbs described in this application provide useful tools in the study of normal B cell development and later stages of erythropoiesis. They may also find applications in the detection, diagnosis, immunophenotyping, classification and prognostication of lymphoid, erythroid and other malignancies in which the malignant cells express penumbra. Such malignancies may include, but not limited to, diffuse large B cell lymphoma (the most common B cell lymphoma), follicular B cell lymphoma, Hodgkin's lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia, acute B lymphocytic leukemia and acute erythroleukemia. Anti-pen mAbs may also be used in combination with other therapeutic modalities such as radiation, surgery, chemotherapy, immunotherapy and biologic response modifiers. Example, anti-pen mAbs may be used together or sequentially with anti-CD20 immunotherapy to increase killing of lymphoma cells while reducing tumor resistance due to reductions in CD20 expression as a result of prior treatment, modulation or mutations. Many autoimmune disorders are caused or accompanied by autoimmune B cells such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, autoimmune hemolytic anemia and idiopathic thrombocytopenia. Thus, anti-pen mAbs may be used to treat autoimmune diseases characterized by disregulated B lymphocytes. About two thirds of transplant patients with an allogeneic bone marrow or hematopoietic progenitor transplant suffer from chronic graft-versus-host disease and many eventually die from it. Chronic graft-versus-host disease is in part caused by “autoimmune” (i.e. from the perspective of the transplant recipients) allogeneic B lymphocytes received or produced from the bone marrow or hematopoietic progenitor grafts. Thus, anti-pen mAbs may find applications in the treatment of chronic graft-versus-host disease as well. Additional usage may include the treatment of B lymphocytes infected with viruses such as Epstein-Barr virus (or cytomegalovirus) as in infectious mononucleosis and post-transplant lymphoproliferative disorder.
While the identification of very low numbers of penLo erythrocytes in blood and/or erythroblasts in bone marrow confirmed our original description of pen mRNA expression in erythroblasts (ref. 3), the detection of this small population may warrant some theoretical consideration in light of the possible application of anti-pen mAbs in the eradication of B cell malignancies such as diffuse large B cell lymphoma, Hodgkin's disease and B cell leukemias as well as B cell-mediated autoimmunity. Although only a very small fraction (estimated to be less than 0.0001%) of erythrocytes and some bone marrow erythroblasts express low levels of pen, it may still be important to reduce the number of such penLo erythrocytes and/or erythroblasts in therapeutic applications of chimeric or humanized anti-hpen mAbs in order to minimize or prevent side effects of hemolysis such as renal toxicity due to the release of free hemoglobin. In practice, most cancer patients already suffer from anemia caused by cancer per se or chemotherapy or both and therefore have rather inactive erythropoiesis with few newly released erythrocytes as reflected in very low reticulocyte counts and the need for red blood cell transfusions. In theory, erythropoiesis can be completely suppressed in such patients by transfusion of red blood cells to a relative high hemoglobin level (e.g. 12-15 g/dL; normal adult males have a hemoglobin level of 14-15 g/dL on average at sea level). High levels of hemoglobin suppress the secretion of erythropoietin, which in turn suppresses erythropoiesis. Thus, we believe that there are safe and effective ways to further reduce the already low number of pent′ erythrocytes should their presence pose any danger to patients receiving Immunotherapy with anti-pen mAbs. In addition, the side effects of hemolysis, if any, can be ameliorated by other measures such as (i) hyperhydration with or without diuretics to encourage a high urine output to reduce the toxicity of free hemoglobin on renal tissues, and (ii) alkalization of urine by intravenous infusion of sodium bicarbonate to prevent precipitation of free hemoglobin in renal tubules. Such measures have already been incorporated into current lymphoma or leukemia chemotherapy to prevent the so-called “tumor lysis syndrome” or in the management of potential transfusion reactions (hemolysis due to ABO or Rh blood type mismatch or anti-donor RBC antibodies). Thus, there already exist several effective measures in the current practice of hematology/oncology clinics to prevent or minimize possible side effects resulting from the interaction between anti-pen mAbs and a small number of penLo erythrocytes. In fact, anti-pen mAbs may be used to treat erythroleukemias that express pen.
Although the description above provides important examples of embodiments, they should not be construed as limiting the scope of the embodiments of this invention but only as illustrations of several embodiments. For example, the chimeric mouse-human anti-pen mAbs may incorporate the constant regions of any human Ig class (IgG, IgM, IgA, IgD, IgE) or subclass (IgG1, IgG2a, IgG2b, IgG3, IgG4, IgA1, IgA2, etc.) or light chain (kappa, lambda). Also, they may be constructed using DNA or amino acid sequences of Igs derived from any species other than mouse and human such as baboon, horse, cow, sheep, camel, llama, dog, cat, rabbit, etc. Furthermore, they are not limited to monovalent, bivalent, multivalent or same-valent antibodies. A single peptide chain of any length or a combination of different peptide chains that retain the antigen-recognition capability of the anti-pen mAbs can be prepared and tested by those skilled in the art. In a further embodiment, the hypervariable-region (or CDR) amino acid sequences of the VRs of anti-pen mAbs can be identified using the IgBLAST software (ncbi.nlm.nih.gov) and grafted onto the FRs of human antibodies to create new mAbs with the same antigen specificity. Such “humanized” antibodies preserve the ability to recognize pen but have a lower potential for triggering allergic reactions in humans. The mAbs disclosed here can also be modified using the well-established method of “conservative amino acid substitution” by those skilled in the art to create mAbs with variant amino acid sequences without significantly impairing the essential pen-binding capacity. In general, alteration of the amino acid sequences outside the CDRs, i.e. in FRs and constant regions, may be made without abolishing the antigen-binding capacity. In addition, any of these molecules can be modified by covalent and noncovalent modifications to alter or impart new properties such as stability (e.g. PEGylation), solubility, body distribution, metabolism, antigenicity, cytotoxicity (e.g. with cytotoxic drugs, predrugs, inhibitors, radioisotopes, complement activators), signaling (e.g. with cytokines, ligands, receptors, enzyme inhibitors or activators), tracking (e.g. with fluorochromes, dyes, enzymes, binding proteins, gold particles, magnetic beads, radiopaque molecules, radioisotopes), etc. using available technology.
This application claims benefit of priority of U.S. Provisional Patent Application Ser. No. 62/391,749 filed May 9, 2016 by the present inventor, the disclosure of which is hereby incorporated by reference in its entirety.
Number | Date | Country | |
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62391749 | May 2016 | US |