CD47 Human, IgG-His

What is the structure and function of CD47, and how does it interact with SIRPα?

CD47 is a transmembrane protein belonging to the immunoglobulin superfamily. It contains an amino-terminal extracellular variable region (IgV-like domain), a transmembrane region composed of highly hydrophobic segments, and a hydrophilic carboxy-terminal cytoplasmic tail that interacts with corresponding ligands . When CD47 binds to SIRPα, it triggers tyrosine phosphorylation on the intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) of SIRPα, which recruits and activates phosphatases including SHP-1 and SHP-2 . This interaction produces a "don't eat me" signal that inhibits macrophage-mediated phagocytosis, protecting cells from immune clearance .

The extracellular domain of CD47 binds to the SIRPα IgV domain, forming a signaling complex that enables cancer cells to escape from macrophage-mediated phagocytosis . This interaction plays a crucial role in immune homeostasis and serves as a mechanism for cells to identify "self" from "non-self" .

How does CD47 expression differ between normal and cancer cells?

CD47 is ubiquitously expressed on the surface of normal cells including red blood cells, platelets, and leukocytes, albeit at varying levels . When normal cells age, such as senescent erythrocytes, CD47 expression decreases, signaling for their clearance by splenic macrophages .

In contrast, CD47 is frequently overexpressed on various cancer cells, including both solid tumors and hematological malignancies . This overexpression correlates with poor patient prognosis in numerous cancer types . By upregulating CD47, cancer cells enhance their ability to evade immune surveillance, as the strengthened CD47-SIRPα interaction inhibits phagocytosis by macrophages . This represents a major immune escape mechanism exploited by tumors.

What expression systems are optimal for producing recombinant CD47 Human, IgG-His proteins?

Multiple expression systems can be used to produce CD47 Human, IgG-His recombinant proteins, with selection depending on research requirements:

For prokaryotic expression, E. coli systems utilizing the pET32a vector in BL21 strains have successfully produced functional CD47-related fusion proteins . Lin et al. constructed recombinant hSIRPext by combining a pET32a plasmid vector with the soluble extracellular domain of SIRPα, which effectively bound to CD47 on leukemia stem cells and blocked CD47-SIRPα signaling .

For applications requiring proper post-translational modifications, mammalian expression systems (CHO or HEK293 cells) are preferred since CD47 is glycosylated. The search results show that various humanized IgG formats (hIgG1, hIgG2, hIgG4) have been successfully used in clinical development, suggesting these formats maintain proper structure and function .

The choice should be guided by the intended application, with mammalian systems preferred when physiologically relevant glycosylation patterns are required for functional studies or therapeutic development.

What are effective methods for validating CD47-SIRPα binding interactions in experimental settings?

Multiple complementary approaches can validate CD47-SIRPα binding:

• Functional phagocytosis assays: Co-incubate CD47 fusion proteins with cancer cells and macrophages to assess inhibition or enhancement of phagocytosis. Lin et al. demonstrated that their CD47 fusion proteins (Trx-hCD47ext and Trx-CD47ext) enhanced the phagocytosis of leukemia cells by macrophages in vitro .

• Flow cytometry: Assess binding of CD47 constructs to SIRPα-expressing cells or vice versa. The humanized CD47/SIRPα knock-in mouse models developed through CRISPR/Cas9 gene-editing were validated using flow cytometry to confirm expression patterns and binding capabilities .

• Competitive binding assays: Use labeled antibodies to measure displacement by test compounds or proteins. Various anti-CD47 antibodies including Hu5F9-G4 have been characterized using such assays .

• In vivo validation: Employ humanized mouse models expressing human CD47 and SIRPα to assess physiological relevance of binding interactions. The research shows that humanized CD47/SIRPα/FcγR mice recapitulate the toxicity observed with CD47-blocking antibodies in clinical settings .

How can humanized mouse models be developed to evaluate CD47-targeting therapeutics?

Development of humanized mouse models for CD47 research involves sophisticated genetic engineering approaches:

Researchers have successfully developed humanized mouse strains in which human extracellular domains (ECD) of CD47 and SIRPα replaced their murine counterparts using CRISPR/Cas9 gene-editing (hCD47/hSIRPα KI mice) . These models express human CD47 ubiquitously on peripheral blood cells (RBCs, platelets, CD3+ and CD11b+ cells) while SIRPα is expressed primarily on CD11b+ cells, accurately recapitulating the human expression pattern .

To improve translational relevance, these mice can be further crossed with FcγR-humanized mice to create triple humanized models (hCD47/hSIRPα/hFcγR) that express all human FcγRs with the human cell-type specific expression profile . These models are particularly valuable because they recapitulate on-target toxicities seen in clinical trials, such as anemia and thrombocytopenia .

Complementary to these whole-animal models, researchers have also generated cancer cell lines (MC38-hCD47 KI, B16-hCD47 KI) with human CD47 ECD replacing the murine counterpart . These cell lines can be engrafted in humanized mouse models to create a comprehensive system for evaluating anti-human CD47 antibodies in vivo .

What strategies can optimize the evaluation of anti-CD47 antibodies with different IgG subclasses and Fc modifications?

The therapeutic index of anti-CD47 antibodies depends significantly on their IgG subclass and Fc region properties:

Clinical trial results indicate that antibodies with weak or null engagement to FcγRs (hIgG4, hIgG2, or inert Fc region) are relatively tolerable but show limited clinical benefit as monotherapy . These antibodies only demonstrate benefit when combined with other tumor-targeting antibodies that engage FcγRs, such as Rituximab or Trastuzumab .

In contrast, antibodies with competent binding to FcγRs (hIgG1 Fc format) show higher therapeutic activity as monotherapy but require lower dosing due to on-target toxicity against normal CD47-expressing cells like RBCs and platelets .

To optimize evaluation, researchers should:

• Compare different antibody formats in parallel using the same experimental systems

• Assess both efficacy (tumor cell phagocytosis) and toxicity (effects on normal cells)

• Utilize humanized mouse models that express human CD47, SIRPα, and FcγRs

• Test specific Fc modifications, such as the GAALIE variant which enhances FcγR engagement

The search results specifically mention comparison between the hIgG4 antibody 5F9 used in clinical trials and its Fc-optimized variant 5F9-GAALIE with enhanced FcγR engagement .

How do different anti-CD47 therapeutic approaches compare in preclinical and clinical settings?

Multiple CD47-targeting approaches have reached clinical trials with varying results:

Fusion proteins: SIRPα-Fc fusion proteins like TTI-621 (Trillium) and ALX148 (Alexo Therapeutics) represent an alternative approach . These proteins bind to CD47 and block its interaction with SIRPα on macrophages.

Combination therapies: Xu et al. demonstrated that combining anti-CD47 antibody with blinatumomab (targeting CD19 and CD3) led to persistent control of lymphoma progression through both enhanced phagocytosis and T-cell cytotoxicity . Similarly, the combination of Hu5F9-G4 and rituximab showed synergistic effects through enhanced macrophage-mediated antibody-dependent phagocytosis in B-cell NHL .

Clinical translation: Phase 1b clinical trials with Hu5F9-G4 and rituximab in patients with DLBCL and follicular lymphoma showed promising results, with adverse events (anemia, nausea, diarrhea, infusion reactions) occurring primarily in the first few weeks without significant safety issues in later stages .

What factors influence CD47's role in cancer metastasis beyond its phagocytosis inhibition function?

CD47 contributes to cancer metastasis through mechanisms independent of its canonical "don't eat me" signal:

Cell migration and invasion: Studies show that targeting CD47 with antibodies or siRNA reduced migration and invasion of cancer cells without necessarily affecting their viability . For example, treating NSCLC cell lines with siRNA to reduce CD47 expression decreased their migration and invasion capabilities without cytotoxicity .

Signaling pathways: CD47 activates downstream effectors such as Cell division control protein 42 (Cdc42), a member of the Rho family of small GTPases that regulates metastasis . In lung cancer cell lines, Cdc42 expression levels positively correlate with CD47 expression . Manipulating Cdc42 levels in CD47-expressing lung cancer cells directly affected their migration and invasion capabilities .

Clinical correlation: In 80 patients with advanced NSCLC, immunohistochemistry revealed a positive correlation between CD47 and Cdc42 expression levels . This suggests that CD47's pro-metastatic function operates at least partly through Cdc42-mediated pathways.

Targeting approaches: Anti-CD47 antibody (B6H12) treatment of osteosarcoma cells reduced their invasiveness and inhibited spontaneous metastasis to the lung in xenograft models while enhancing phagocytosis by macrophages .

What emerging combination strategies with CD47 blockade show the most promise for cancer immunotherapy?

Several combination approaches are being explored to enhance the efficacy of CD47-targeting therapies:

Anti-CD47 antibodies with tumor-targeting antibodies: The combination of Hu5F9-G4 (anti-CD47) with rituximab (anti-CD20) has shown synergistic effects in B-cell NHL by enhancing macrophage-mediated antibody-dependent cellular phagocytosis . This approach leverages both the blockade of the "don't eat me" signal and the provision of a "eat me" signal through Fc receptor engagement.

Anti-CD47 with T-cell engagers: Xu et al. demonstrated that combining anti-CD47 antibody with blinatumomab (CD19/CD3 bispecific antibody) led to persistent control of lymphoma progression by simultaneously enhancing macrophage phagocytosis and T-cell cytotoxicity . This approach engages both innate and adaptive immune mechanisms.

Optimized antibody engineering: Comparing different antibody formats (IgG1 vs. IgG4) and Fc modifications (like GAALIE) can improve the therapeutic index . Antibodies with enhanced FcγR engagement show better therapeutic activity as monotherapy but require careful dosing to manage on-target toxicity .

Development of bispecific antibodies that simultaneously target CD47 and tumor-specific antigens represents another promising direction to enhance specificity and reduce off-target effects.

How can CD47 research models be improved to better predict clinical outcomes of CD47-targeting therapies?

Current models have limitations in predicting clinical outcomes, but several improvements are being developed:

Triple-humanized mouse models: The development of mice humanized for CD47, SIRPα, and FcγRs represents a significant advancement . These models more accurately recapitulate the toxicities observed in clinical settings, such as anemia and thrombocytopenia . They overcome limitations of xenograft models in immunocompromised mice that fail to capture on-target off-tumor effects .

Cancer cell line engineering: Creating cancer cell lines with human CD47 extracellular domains (MC38-hCD47 KI, B16-hCD47 KI) allows for more relevant tumor models when engrafted in humanized mice . This approach maintains an intact immune system while enabling evaluation of human-specific anti-CD47 antibodies.

Patient-derived organoids and xenografts: Incorporating patient samples into 3D culture systems or as xenografts in humanized mice could better represent tumor heterogeneity and the complex tumor microenvironment.

Multi-parameter assessment: Future models should simultaneously evaluate efficacy (tumor control), toxicity (effects on normal cells), pharmacokinetics, and immune activation to provide a comprehensive view of therapeutic potential.

Incorporating these advanced models into preclinical development pipelines would likely improve the translation of promising CD47-targeting therapies to successful clinical outcomes.

Human Experimentation: CD47 and SIRPα