Antibodies and immunoconjugates and uses thereof
An immunoconjugate, antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulin, immunoglobulin, etc., can solve the problem of low efficacy
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Embodiment 1
[1238] Example 1: Preparation of mouse anti-human CD22 monoclonal antibody
[1239] A murine monoclonal antibody capable of specifically binding to human CD22 was prepared. Six-week-old BALB / c female mice were immunized in their paw pads with purified human CD22 plus his-8-tagged extracellular domain lacking domains 3 and 4 in Ribi's adjuvant (SEQ ID NO: 30 (ECD) at the C-terminus plus the sequence GRAHHHHHHHH) or the extracellular domain of CD22 plus his-8 tag comprising domains 1-7 (SEQ ID NO: 28 (ECD) plus the above His sequence tag). Subsequent injections were administered in the same manner one and three weeks after the initial immunization. Three days after the final injection, inguinal and popliteal lymph nodes were removed and pooled, and single cell suspensions were prepared by passing the tissue through a steel gauge. The cells were fused with mouse myeloma, such as P3X63-Ag8.653 (ATCC CRL 1580), at a 4:1 ratio in high glucose (DMEM) containing 50% w / v polyethylene...
Embodiment 2
[1242] Example 2: FACS-based assay for the analysis of anti-human CD22 monoclonal antibodies (MAbs)
[1243] CHO cells expressing human CD22 on their surface were incubated with anti-CD22 hybridoma supernatant in 100 μl FACS buffer (0.1% BSA in PBS, pH 7.4, 10 mM sodium azide) at 4° C. for 30 minutes, Then wash once with FACS buffer. The amount of anti-CD22 binding was determined by aliquots of the antibody / cell mixture with polyclonal FITC-conjugated goat or rabbit anti-mouse IgG (Accurate Chem. Co., Westbury, NY) (for mouse testing antibody) or goat or rabbit anti-human IgG (for humanized antibodies) were incubated at 4°C for 30 minutes, followed by three washes with FACS buffer.
Embodiment 3
[1244] Example 3: Preparation of humanized anti-CD22 antibody
[1245] A humanized 10F4 antibody was generated in which hypervariable region (HVR) amino acid residues (interchangeably referred to as complementarity determining region or CDR) to obtain two variants, humanized 10F4v1 and humanized 10F4v2 (also referred to herein as "10F4v1" or "hu10F4v1" or "10F4v2" or "hu10F4v2", respectively). A third version, humanized 10F4v3 ("10F4v3" or "hu10Fv3") used in some of the studies disclosed herein, has the same mature protein light and heavy chain amino acid sequences as The expression vectors contain different signal sequences.
[1246] Humanization of the murine 10F4 antibody was performed as disclosed herein. Briefly, the light and heavy chain hypervariable regions of murine 10F4 were cloned into a modified consensus framework sequence to generate Figure 2A with 2B Amino acid sequences of light and heavy chain variable regions shown in . Alternative light and heavy chain...
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