Figure S1: IdeS expression in BT474 breast cancer cells Figure S2: Detection of MMPs in cancer cell lysates and culture supernatant Figure S3: scIgG-T was not in the SKBR-3 cell culture medium Figure S4: Detection of immune cells infiltration in mice by flow cytometry.

Supplementary Tables 1-2, Supplementary Figures 1-6. Supplemental table 1. Patient clinical information; Supplemental table 2 Patient clinical information; Supplemental Figure 1. Standard curves developed for calculation of IgG subclasses; Supplemental Figure 2. Profiles of IgG subclasses in tumor and plasma samples; Supplemental Figure 3. IgG2 subclass showed much lower binding to Fc gamma receptors than the IgG1 counterpart; Supplemental Figure 4. Elevated scIgGs in tumor tissues were associated with reduced interactions with Fc gamma receptors; Supplemental Figure 5. Breast tumor tissues had significantly higher sum total of MMPs (MMP-1, 2 , 3, 8, 9, 10, 13) than in normal breast tissues; Supplemental Figure 6. There was no clear correlation between scIgG levels in tumor tissues and patient cancer grades or stages at the time of diagnosis

Natural killer (NK) cells mediate antibody dependent cytotoxic killing of cancer cells via cross-linking FcγR on NK cells with IgG-Fc. Studies have shown that the single-hinge cleaved IgGs (scIgGs) have dysfunctional Fc and failed engagement with FcγRs on immune cells. However, little is known about how scIgGs impact on antitumor immunity in the tumor microenvironment. In this study, we revealed a significant association of tumor scIgGs with tumor progression and poor outcomes of breast cancer patients ( n = 547). Using multiple mouse tumor models, we demonstrated that tumor scIgGs reduced NK cell cytotoxic activities and resulted in aggressive tumor progression. We further showed that an anti-hinge specific monoclonal antibody (AHA) rescued the dysfunctional Fc in scIgGs by providing a functional Fc and restored NK cell cytotoxic activity. These findings point to a novel immunotherapeutic strategy to enhance Fc engagement with FcγRs for activation of anticancer immunity.

BACKGROUND:Proteolytic impairment of the Fc effector functions of therapeutic monoclonal antibodies (mAbs) can compromise their antitumor efficacy in the tumor microenvironment and may represent an unappreciated mechanism of host immune evasion. Pertuzumab is a human epidermal growth factor receptor 2 (HER2)-targeting antibody and has been widely used in the clinic in combination with trastuzumab for treatment of HER2-overexpressing breast cancer. Pertuzumab susceptibility to proteolytic hinge cleavage and its impact on the drug's efficacy has not been previously studied.METHODS:Pertuzumab was incubated with high and low HER2-expressing cancer cells and proteolytic cleavage in the lower hinge region was detected by western blotting. The single hinge cleaved pertuzumab (scIgG-P) was purified and evaluated for its ability to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro and anti-tumor efficacy in vivo. To assess the cleavage of trastuzumab (IgG-T) and pertuzumab (IgG-P) when simultaneously bound to the same cancer cell surface, F(ab')2 fragments of IgG-T or IgG-P were combined with the intact IgG-P and IgG-T, respectively, to detect scIgG generation by western blotting.RESULTS:Pertuzumab hinge cleavage occurred when the mAb was incubated with high HER2-expressing cancer cells. The hinge cleavage of pertuzumab caused a substantial loss of ADCC in vitro and reduced antitumor efficacy in vivo. The reduced ADCC function of scIgG-P was restored by an anti-hinge mAb specific for a cleavage site neoepitope. In addition, we constructed a protease-resistant version of the anti-hinge mAb that restored ADCC and the cell-killing functions of pertuzumab when cancer cells exressed a potent IgG hinge-cleaving protease. We also observed increased hinge cleavage of pertuzumab when combined with trastuzumab.CONCLUSION:The reduced Fc effector function of single hinge-cleaved pertuzumab can be restored by an anti-hinge mAb. The restoration effect indicated that immune function could be readily augmented when the damaged primary antibodies were bound to cancer cell surfaces. The anti-hinge mAb also restored Fc effector function to the mixture of proteolytically disabled trastuzumab and pertuzumab, suggesting a general therapeutic strategy to restore the immune effector function to protease-inactivated anticancer antibodies in the tumor microenvironment. The findings point to a novel tactic for developing breast cancer immunotherapy.

The host immune system adopts multiple mechanisms involving antibodies to confront cancer cells. Accordingly, anti-tumor mAbs have become mainstays in cancer treatment. However, neither host immunity nor mAb therapies appear capable of controlling tumor growth in all cases. Structural instability of IgG was overlooked as a factor contributing to immunosuppression in the tumor microenvironment. Recently, physiological proteinases were identified that disable IgG immune effector functions. Evidence shows that these proteinases cause localized IgG impairment by selective cleavage of a single IgG peptide bond in the hinge-region. The recognition of IgG cleavage in the tumor microenvironment provides alternatives for tumor immunotherapy.

Pathogens that induce acute and chronic infections, as well as certain cancers, employ numerous strategies to thwart host cellular and humoral immune defenses. One proposed evasion mechanism against humoral immunity is a localized expression of extracellular proteases that cleave the IgG hinge and disable host IgG functions. Host immunity appears to be prepared to counter such a proteolytic tactic by providing a group of autoantibodies, denoted anti-hinge antibodies that specifically bind to cleaved IgGs and provide compensating functional restoration in vitro. These respective counter-measures highlight the complex interrelationships among pathogens and host immunity and suggested to us a possible means for therapeutic intervention. In this study, we combined an investigation of pathogen-mediated proteolysis of host IgGs with an immunization strategy to boost host anti-hinge antibodies. In a Staphylococcus aureus infection model using an artificial tissue cage (wiffle ball) implanted into rabbits, cleaved rabbit IgGs were detected in abundance in the abscesses of untreated animals early after infection. However, in animals previously immunized with peptide analogs of the cleaved IgG hinge to generate substantial anti-hinge antibody titers, S. aureus colony formation was markedly reduced compared to control animals or those similarly immunized with a scrambled peptide sequence. The results of this study demonstrate that extensive local proteolysis of IgGs occurs in a test abscess setting and that immunization to increase host anti-hinge antibodies provided substantial acute protection against bacterial growth.

Immune suppression is recognized as a hallmark of cancer and this notion is largely based on studies on cellular immunity. Our recent studies have demonstrated a potential new mechanism of cancer suppression of immunity by impairment of antibody effector function mediated by proteolytic enzymes in the tumor microenvironment.

Abstract Antibodies can elicit multiple functions through its Fc region, including cell-killing through antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxity (CDC) and antibody-dependent cellular phagocytosis (ADCP). In addition to the cell-killing functions, antibodies also mediate antibody-dependent cytokine release (ADCR). In this study, we demonstrate that ADCR is regulated differentially in PBMC and macrophages. ADCR is FcγRIII-dependent in PBMC and mediated by NK cells. In contrast, ADCR in macrophages is FcγRI-dependent. In PBMC, ADCR is characterized by expression of primarily pro-inflammatory cytokines including IFN-γ, IL-1α, IL-1ß, TNF-α. Meanwhile in macrophages, ADCR results in a mixture of the anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines including IFN-γ, IL-1α, IL-1ß, TNF-α, and RANTES. Using an Fc engineering approach, we generated antibody variants lacking FcγRI binding while maintaining binding to FcγRIIa and FcγRIII. These variants retain ADCC and ADCR in PBMC, but modulate ADCP without eliciting cytokine release in macrophages, separating the two processes. Ultimately, a better understanding of how cytokine release is regulated by antibodies and of strategies to modulate cytokine release through Fc engineering could be used to influence immune cell fate decisions in vivo. Citation Format: Michelle Kinder, Allison Greenplate, William Strohl, Robert Jordan, Randall Brezski. Modulation of antibody-dependent cytokine release using Fc engineering. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 638. doi:10.1158/1538-7445.AM2015-638

BACKGROUND & AIMS:Many patients with inflammatory bowel disease (IBD) fail to respond to anti-tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn's disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNF-neutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs.METHODS:Biopsy specimens from inflamed colon of 8 patients with Crohn's disease and 8 patients with ulcerative colitis, and from normal colon of 8 healthy individuals (controls), were analyzed histologically, or homogenized and proteins were extracted. We also analyzed sera from 29 patients with active Crohn's disease and 33 patients with active ulcerative colitis who were candidates to receive infliximab treatment. Infliximab, adalimumab, and etanercept were incubated with mucosal homogenates from patients with IBD or activated recombinant human MMP3 or MMP12 and analyzed on immunoblots or in luciferase reporter assays designed to measure TNF activity. IgG cleaved by MMP3 or MMP12 and antihinge autoantibodies against neo-epitopes on cleaved IgG were measured in sera from IBD patients who subsequently responded (clinical remission and complete mucosal healing) or did not respond to infliximab.RESULTS:MMP3 and MMP12 cleaved infliximab, adalimumab, and etanercept, releasing a 32-kilodalton Fc monomer. After MMP degradation, infliximab and adalimumab functioned as F(ab')2 fragments, whereas cleaved etanercept lost its ability to neutralize TNF. Proteins from the mucosa of patients with IBD reduced the integrity and function of infliximab, adalimumab, and etanercept. TNF-neutralizing function was restored after incubation of the drugs with MMP inhibitors. Serum levels of endogenous IgG cleaved by MMP3 and MMP12, and antihinge autoantibodies against neo-epitopes of cleaved IgG, were higher in patients who did not respond to treatment vs responders.CONCLUSIONS:Proteolytic degradation may contribute to the nonresponsiveness of patients with IBD to anti-TNF agents.

Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.

Trastuzumab has been used for the treatment of HER2-overexpressing breast cancer for more than a decade, but the mechanisms of action for the therapy are still being actively investigated. Ab-dependent cell-mediated cytotoxicity mediated by NK cells is well recognized as one of the key mechanisms of action for trastuzumab, but trastuzumab-mediated Ab-dependent cellular phagocytosis (ADCP) has not been established. In this study, we demonstrate that macrophages, by way of phagocytic engulfment, can mediate ADCP and cancer cell killing in the presence of trastuzumab. Increased infiltration of macrophages in the tumor tissue was associated with enhanced efficacy of trastuzumab whereas depletion of macrophages resulted in reduced antitumor efficacy in mouse xenograft tumor models. Among the four mouse FcγRs, FcγRIV exhibits the strongest binding affinity to trastuzumab. Knockdown of FcγRIV in mouse macrophages reduced cancer cell killing and ADCP activity triggered by trastuzumab. Consistently, an upregulation of FcγRIV expression by IFN-γ triggered an increased ADCP activity by trastuzumab. In an analogous fashion, IFN-γ priming of human macrophages increased the expression of FcγRIII, the ortholog of murine FcγRIV, and increased trastuzumab-mediated cancer cell killing. Thus, in two independent systems, the results indicated that activation of macrophages in combination with trastuzumab can serve as a therapeutic strategy for treating high HER2 breast cancer by boosting ADCP killing of cancer cells.

Abstract Purpose: Studies have demonstrated that cancer-associated matrix metalloproteinases (MMP) can generate single peptide bond cleavages in the hinge region of immunoglobulin G1 (IgG1). This study investigated the cleavage of endogenous IgGs by MMPs in the tumor microenvironment and the consequences of the IgG hinge cleavage for humoral immunity. Experimental Design: We investigated the occurrence of single peptide bond cleaved IgGs (scIgG) in tumor tissues and plasma samples collected from a cohort of breast cancer patients (n = 60). Samples from healthy people (n = 20) were used as the control. Antibody hinge cleavage was detected by multiple assays, including IHC, ELISA, and flow cytometry. A correlation analysis was conducted between scIgG levels and patient clinical parameters. Results: Levels of scIgGs in tumors were significantly higher than in normal tissues. In addition, scIgG levels in tumors were enriched compared with that in the plasma of the same patients. The appearance of scIgGs in tumor tissues was associated with altered host IgG content and decreased IgG1. Increased tumor scIgGs were found to be positively correlated with adverse clinical factors, such as elevated tumor-associated macrophages, increased expression of MMP9 and other MMPs, and local metastasis to axillary lymph nodes. Conclusions: The study contributes to mounting evidence for the presence of hinge-cleaved antibodies with reduced Fc immune effector function in the tumor microenvironment. The results highlight a link between tumor scIgGs and poor patient outcomes, and reveal a component of compromised humoral immunity within tumors that could point to new immunotherapeutic strategies to rescue host immunity. Clin Cancer Res; 21(23); 5380–90. ©2015 AACR.

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab')₂ fragments, but not for full-length IgG or for closely-related F(ab')₂ fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab')₂ fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab')₂ fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab')₂ fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab')₂ fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab')₂ fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab')₂, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.

The functional role of human antihinge (HAH) autoantibodies in normal health and disease remains elusive, but recent evidence supports their role in the host response to IgG cleavage by proteases that are prevalent in certain disorders. Characterization and potential exploitation of these HAH antibodies has been hindered by the absence of monoclonal reagents. 2095-2 is a rabbit monoclonal antibody targeting the IdeS-cleaved hinge of human IgG1. We have determined the crystal structure of the Fab of 2095-2 and its complex with a hinge analog peptide. The antibody is selective for the C-terminally cleaved hinge ending in G236 and this interaction involves an uncommon disulfide in VL CDR3. We probed the importance of the disulfide in VL CDR3 through engineering variants. We identified one variant, QAA, which does not require the disulfide for biological activity or peptide binding. The structure of this variant offers a starting point for further engineering of 2095-2 with the same specificity, but lacking the potential manufacturing liability of an additional disulfide. Proteins 2014; 82:1656-1667. © 2014 Wiley Periodicals, Inc.

AbstractPrimary and acquired resistance to anticancer antibody immunotherapies presents significant clinical challenges. Here, we demonstrate that proteolytic inactivation of cancer-targeting antibodies is an unappreciated contributor to cancer immune evasion, and the finding presents novel opportunities for therapeutic intervention. A single peptide bond cleavage in the IgG1 hinge impairs cancer cell killing due to structural derangement of the Fc region. Hinge-cleaved trastuzumab gradually accumulated on the surfaces of HER2-expressing cancer cell lines in vitro, and was greatly accelerated when the cells were engineered to express the potent bacterial IgG-degrading proteinase (IdeS). Similar to cancer-related matrix metalloproteinases (MMP), IdeS exposes a hinge neoepitope that we have developed an antibody, mAb2095-2, to specifically target the epitope. In in vitro studies, mAb2095-2 restored the lost antibody-dependent cell-mediated cytotoxicity functionality of cell-bound single-cleaved trastuzumab (scIgG-T). In vivo, mAb2095-2 rescued the impaired Fc-dependent tumor-suppressive activity of scIgG-T in a xenograft tumor model and restored the recruitment of immune effector cells into the tumor microenvironment. More importantly, an Fc-engineered proteinase-resistant version of mAb2095-2 rescued trastuzumab antitumor efficacy in a mouse tumor model with human cancer cells secreting IdeS, whereas trastuzumab alone showed significantly reduced antitumor activity in the same model. Consistently, an Fc-engineered proteinase-resistant version of trastuzumab also greatly improved antitumor efficacy in the xenograft tumor model. Taken together, these findings point to a novel cancer therapeutic strategy to rescue proteolytic damage of antibody effector function by an Fc-engineered mAb against the hinge neoepitope and to overcome cancer evasion of antibody immunity. Mol Cancer Ther; 14(3); 681–91. ©2014 AACR.

Invasive cancers express matrix metalloproteases, such as MMP-3 and MMP-7, which have the ability to cleave human antibodies in the lower hinge region. The human IgG lower hinge region contains two heavy chains, and cleavage occurs in a two-step process. First, one heavy chain is cleaved, resulting in a singly-cleaved intermediate. Cleavage of the second heavy chain liberates the Fc region from the antigen-binding region, resulting in an Fc fragment and a F(ab’)2 fragment. Previous work has demonstrated that the singly-cleaved intermediate accumulates on the surface of IgG1-opsonzied tumor cells in the presence of MMPs. The singly-cleaved intermediate is indistinguishable from the intact IgG counterpart when assessed by analytical techniques under native conditions and maintains normal antigen binding. However, the singly-cleaved intermediate displays a profound loss of Fc-mediated cell-killing functions both in vitro (e.g. ADCC and CDC) and with in vivo cell clearance, presumably because the lower hinge contains key recognition points for both FcγRs and the C1q component of complement. Singly-cleaved IgGs are of particular concern with regards to human IgG1 monoclonal therapeutic antibodies, because the proposed mechanisms of action for several anti-tumor mAbs in the clinic may be partially dependent on Fc effector functions, such as ADCC and CDC. For this reason, our group and others have proposed that cleavage and the subsequent inactivation of Fc-effector functions of human IgG antibodies may function as an immune evasion mechanism. To overcome the loss-of-function associated with human IgG1 cleavage, we aimed to develop a protease-resistant IgG1 platform which would still maintain Fc-mediated cell-killing functions. Mutation of the lower hinge of IgG1 provided protease-resistance, but also resulted in a loss of Fc-effector functions. Through specific mutations of amino acids in the CH2 region to enhance complement or Fcγ receptor binding, we were able restore or even enhance cell-killing functions selectively while maintaining protease-resistance. Therefore, we were able to generate protease-resistant antibodies with the ability to mediate either complement-dependent and/or antibody-dependent cellular cytotoxicity, which were also significantly resistant to inactivation by proteases associated with many invasive cancers. Citation Format: Michelle Kinder, Katharine Grugan, Keri Soring, William Strohl, Robert Jordan, Randall Brezski. A novel protease resistant antibody platform with selectable cell killing functions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4327. doi:10.1158/1538-7445.AM2013-4327

Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the CH2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality.

INTRODUCTION:Recent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model.METHODS:Trastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry.RESULTS:scIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro.CONCLUSION:Trastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where matrix metalloproteinases often have high levels of expression and scIgG-T might compromise its anti-tumor efficacy in the clinic. However, further studies are needed to validate these hypotheses in the clinical setting.