Patent Application
20230332245
The present invention relates in general to the field of biomarkers, and more particularly, to Complement as prognostic and predictive biomarker and potential therapeutic target in renal cell carcinoma.
The present application includes a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 28, 2021, is named TECH2153WO_ST25.txt and is 5, kilo bytes in size.
Without limiting the scope of the invention, its background is described in connection with renal cell carcinoma.
Early studies demonstrated deposition of complement proteins in several human malignancies [1]. Complement was thought to contribute to immune surveillance through complement dependent cytotoxicity (CDC) and tumor cell lysis [2]. These roles for complement are particularly clear in the context of anticancer monoclonal antibody therapies, where CDC assists in tumor cell killing, especially in hematologic malignancies [3]. However, because of overexpression of membrane and soluble complement regulatory (i.e. inhibitory) proteins, solid tumors are protected from complement-mediated lysis [2]. Consequently, the functional significance of complement activation in tumors in the absence of therapeutic antibodies has remained unclear.
In contrast to these beneficial antitumor complement roles, several studies demonstrated that the complement system promotes tumor growth by inhibiting antitumor immunity [4-6]. In fact, complement is currently perceived as an important immunosuppressive mechanism in primary tumors [6, 7] and metastasis-targeted organs [8, 9]. Complement proteins activate and recruit immunosuppressive cells, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), and regulatory T cells (Tregs), to tumors and premetastatic niches [7, 10]. Recent work also demonstrated synergism between programmed cell death protein 1 (PD-1) blockade and complement inhibition to reduce tumor growth [11]. Interestingly, the C5a/C5a receptor 1 (C5aR1) axis was shown to have prognostic value in human renal cell carcinoma (RCC) [12]. In addition, the C1q and the classical pathway appear to contribute to RCC progression [13]. However, despite this foundational knowledge, complement-based anticancer therapies have not yet advanced to the clinic [14]. This may be due to two factors: (i) lack of understanding of best therapeutic targets within the complement cascade; and (ii) lack of knowledge about which cancer patients might benefit from complement-based therapies.
However, despite these advancements, a need remains for improved predictability and reliability of biomarkers, and methods of treatment for renal cell carcinomas.
In one embodiment, the present invention includes a method of determining a prognosis of a subject with cancer comprising: obtaining or having obtained a sample from the subject; and measuring in the sample a level of expression of one or more Complement or Complement related genes or proteins; and determining if the levels of expression of the Complement or Complement related gene or protein when compared to the levels of expression of the Complement or Complement related genes or proteins from a subject that does not have cancer, wherein a change in the level of expression of the Complement or Complement related genes or proteins is associated with an unfavorable prognosis or a favorable prognosis. In one aspect, the cancer is selected from renal, urothelial, stomach, liver, pancreatic, breast, head/neck, testis, ovarian, and cervical. In another aspect, the Complement or Complement related gene or protein is selected from C1QA, C1QB, C1S, C1R, C2, C3, C5, C6, C7, CSB, CFB, CFD, CFH, CFI, CD21/CR2, CD46, CD55, CD59, C5AR1. In another aspect, the Complement or Complement related gene or protein is favorable and is selected from at least one of:
In another aspect, the Complement or Complement related gene or protein is unfavorable and is selected from at least one of:
In another aspect, a histological grade of the cancer is determined by the expressed or deposition of Complement proteins in tumor stroma. In another aspect, the Complement or Complement related gene or protein CFB, C5AR1, CFH, C3, C1R, CIS C1QA, and C1QB are enriched in aggressive inflammatory phenotype cancers. In another aspect, the method further comprises determining a level of expression of macrophage biomarkers selected from CD86, IRF1, STAB1; TFGB1, F13A1, IL-6, and CD40, wherein expression of one or more of the macrophage biomarkers is associated with an unfavorable prognosis. In another aspect, the sample is a plasma sample. In another aspect, the method further comprises separating a subject into a those with a higher or a lower level of expression of the Complement or Complement related gene or protein, and: if the subject has low FH and FD expression the subject has a worse response to an immune checkpoint inhibitor; if the subject has low FI and TCC the subject has a better response to an immune checkpoint inhibitor; or if the subject has low TCC and high C5 the subject has a better response to an immune checkpoint inhibitor. In another aspect, the immune checkpoint inhibitor is selected from nivolumab, ipilimumab, tremelimumab, ipilimumab and nivolumab, pembrolizumab, nivolumab, pidilizumab, MK-3475, MED 14736, CT-011, spartalizumab, durvalumab, atezolizumab, avelumab, AMP224, BMS-936559, MPLDL3280A, or MSB0010718C. In another aspect, the immune checkpoint inhibitor is selected from inhibitors of at least one of: CD137, CD134, PD-1, KIR, LAG-3, PD-L1, PDL2, CTLA-4, B7.1, B7.2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, BTLA, LIGHT, HVEM, GAL9, TIM-3, TIGHT, VISTA, 2B4, CGEN-15049, CHK 1, CHK2, A2aR, TGF-beta, PI3Kgamma, GITR, ICOS, IDO, TLR, IL-2R, IL-10, PVRIG, CCRY, OX-40, CD160, CD20, CD52, CD47, CD73, CD27-CD70, or CD40. In another aspect, the method further comprises the step of treating a renal cell carcinoma with treated with C3aR1 and C5aR1 inhibitors to reduce tumor growth. In another aspect, the method further comprises the step of treating the subject with a complement blockade to at least one of: reduced vascular density in tumors or reduced expression of proangiogenic factors.
In another embodiment, the present invention includes a method of treating a subject with cancer comprising: obtaining or having obtained a sample from the subject; and measuring in the sample a level of expression of one or more Complement or Complement related genes or proteins; determining if the levels of expression of the Complement or Complement related gene or protein when compared to the levels of expression of the Complement or Complement related genes or proteins from a subject that does not have cancer, wherein a change in the level of expression of the Complement or Complement related genes or proteins is associated with an unfavorable prognosis or a favorable prognosis; and if the subject has low FH and FD expression the subject has a worse response to an immune checkpoint inhibitor; if the subject has low FI and TCC the subject has a better response to an immune checkpoint inhibitor; or if the subject has low TCC and high C5 the subject has a better response to an immune checkpoint inhibitor. In one aspect, the immune checkpoint inhibitor is selected from nivolumab, ipilimumab, tremelimumab, ipilimumab and nivolumab, pembrolizumab, nivolumab, pidilizumab, MK-3475, MED 14736, CT-011, spartalizumab, durvalumab, atezolizumab, avelumab, AMP224, BMS-936559, MPLDL3280A, or MSB0010718C. In another aspect, the immune checkpoint inhibitor is selected from inhibitors of at least one of: CD137, CD134, PD-1, KIR, LAG-3, PD-L1, PDL2, CTLA-4, B7.1, B7.2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, BTLA, LIGHT, HVEM, GAL9, TIM-3, TIGHT, VISTA, 2B4, CGEN-15049, CHK 1, CHK2, A2aR, TGF-beta, PI3Kgamma, GITR, ICOS, IDO, TLR, IL-2R, IL-10, PVRIG, CCRY, OX-40, CD160, CD20, CD52, CD47, CD73, CD27-CD70, or CD40. In another aspect, the cancer is selected from renal, urothelial, stomach, liver, pancreatic, breast, head/neck, testis, ovarian, and cervical. In another aspect, a histological grade of the cancer is determined by the expressed or deposition of Complement proteins in tumor stroma. In another aspect, the Complement or Complement related gene or protein CFB, C5AR1, CFH, C3, C1R, CIS C1QA, and C1QB are enriched in aggressive inflammatory phenotype cancers. In another aspect, the method further comprises determining a level of expression of macrophage biomarkers selected from CD86, IRF1, STAB1, TFGB1, F13A1, IL-6, and CD40, wherein expression of one or more of the macrophage biomarkers is associated with an unfavorable prognosis. In another aspect, the sample is a plasma sample. In another aspect, the method further comprises the step of treating a renal cell carcinoma with treated with C3aR1 and C5aR1 inhibitors to reduce tumor growth. In another aspect, the method further comprises the step of treating the subject with a complement blockade to at least one of: reduced vascular density in tumors or reduced expression of proangiogenic factors.
In another embodiment, the present invention includes a method for treating a cancer comprising the steps of: performing or having performed a level of expression of one or more Complement or Complement related genes or proteins; determining if the levels of expression of the Complement or Complement related gene or protein when compared to the levels of expression of the Complement or Complement related genes or proteins from a subject that does not have cancer, wherein a change in the level of expression of the Complement or Complement related genes or proteins is associated with an unfavorable prognosis or a favorable prognosis; and if the subject has low FH and FD expression the subject has a worse response to an immune checkpoint inhibitor; if the subject has low FI and TCC the subject has a better response to an immune checkpoint inhibitor; or if the subject has low TCC and high C5 the subject has a better response to an immune checkpoint inhibitor. In one aspect, the immune checkpoint inhibitor is selected from nivolumab, ipilimumab, tremelimumab, ipilimumab and nivolumab, pembrolizumab, nivolumab, pidilizumab, MK-3475, MED 14736, CT-011, spartalizumab, durvalumab, atezolizumab, avelumab, AMP224, BMS-936559, MPLDL3280A, or MSB0010718C. In one aspect, the immune checkpoint inhibitor is selected from inhibitors of at least one of: CD137, CD134, PD-1, KIR, LAG-3, PD-L1, PDL2, CTLA-4, B7.1, B7.2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, BTLA, LIGHT, HVEM, GAL9, TIM-3, TIGHT, VISTA, 2B4, CGEN-15049, CHK 1, CHK2, A2aR, TGF-beta, PI3Kgamma, GITR, ICOS, IDO, TLR, IL-2R, IL-10, PVRIG, CCRY, OX-40, CD160, CD20, CD52, CD47, CD73, CD27-CD70, or CD40.
For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
The present inventors performed a systematic analysis of expression of several complement genes in human solid tumors to identify: (1) cancer patients with deregulated complement that might potentially benefit from complement-based interventions and (2) complement-dependent mechanisms regulating tumor growth that can be targeted for therapy. The inventors complemented these studies with comprehensive analyses of complement proteins in plasma and investigated their predictive potential for the response to immune checkpoints inhibitors (ICI). Findings in patients were corroborated in a mouse model of RCC.
The present inventors determined the best target for complement-based therapy amongst common human malignancies. High expression of eleven complement genes was linked to unfavorable prognosis in renal cell carcinoma. Complement protein expression or deposition was observed mainly in stroma, leukocytes, and tumor vasculature, corresponding to a role of complement in regulating the tumor microenvironment. Complement abundance in tumors correlated with a high nuclear grade. Complement genes clustered within an aggressive inflammatory subtype of renal cancer characterized by poor prognosis, markers of T cell dysfunction, and alternatively activated macrophages. Plasma levels of complement proteins correlated with response to immune checkpoint inhibitors. Corroborating human data, complement deficiencies and blockade reduced tumor growth by enhancing antitumor immunity and seemingly reducing angiogenesis in a mouse model of kidney cancer-resistant to PD-1 blockade. Thus, tumors resistant to immune checkpoint inhibitors are suitable targets for complement-based therapy. By identifying patients with the optimal biomarker profile of complement proteins who will respond to immune checkpoint inhibitors, healthcare providers will now be able to offer better survival outcomes, minimize adverse reactions and invasive procedures, and reduce costs for patients and insurance companies. As a result, present invention provides a less invasive, faster, and offers improved diagnostics for all patients.
Human Samples and Data
Data on complement genes' RNA expression and survival were obtained from Human Protein Atlas (https://www.proteinatlas.org/) and the Cancer Genome Atlas (https://cancergenome.nih.gov/) or from University of Texas Southwestern Medical Center Kidney Cancer Program (UTSW KCP) as previously reported [15]. The p values included in the Table I and
Data Availability
Sequencing data from UTSW KCP patients, specifically consenting to placement of their raw genomic data in a protected publicly accessible database, are deposited in the European Genome—phenome Archive (EGA) (https://www.ebi.ac.uk/ega/home), with accession numbers EGAS00001002786 and EGAS00001000926.
Mice, Cell Lines, and Treatments
Mouse studies were approved by the Institutional Animal Care and Use Committee of the Texas Tech University Health Sciences Center. Eight to twelve weeks old BALB/c, C3ar1, and C5ar1 knockout mice from the Jackson Laboratory were injected s.c. with 1×106 Renca cells (ATCC® CRL2947™). When tumors reached ˜5 mm in diameter mice were assigned to treatment cohorts and treated with, C3aR1 inhibitor SB 290157 (Sigma-Aldrich, 10 mg/kg i.p. twice a day), C5aR1 inhibitor PMX53 [17] (1 mg/kg, i.p. every other day), or PD-1 antibody (RMP1-14, BE-0146, Bio X Cell, 250 μg per mouse i.p. every 4 days), or RaIgG2a isotype control (BE-0089, Bio X Cell, 250 μg i.p. every 4 days), as previously described [8, 18, 19]. CD8+ T cells were depleted by intraperitoneal (i.p.) injection of 200 μg of CD8a-neutralizing antibody (2.43, Bio X cell, NH) per mouse, each day, for three consecutive days, prior to injecting tumor cells. To maintain CD8+ T cell depletion, mice were injected with 200 μg of antibody every 3rd day beginning at day 3 after tumor cell injection. BALB/c control mice were treated in the same manner with rat IgG2b (LTF-2, Bio X cell).
Immunofluorescence
5-μm thick frozen tissue sections were stained with CD31 (Clone 390, BD Pharmingen), CD8α (53-6.7, BD Pharmingen), CD11b (550282, BD Biosciences), CD88(C5aR1) (sc-31240, Santa Cruz Biotechnology), C3b/iC3b/C3c, which binds only to C3 cleavage fragments but not to intact C3 [20] (HM 1065, Hycult Biotech), C1q (HM 1096BT, Hycult), mannan binding lectin (NB100-1502 Novus), IgM antibodies (14-5790-81, eBioscience) and Annexin V (sc-1929, Santa Cruz Biotechnology). Secondary antibodies included: Goat anti-rat antibodies (Invitrogen): Streptavidin-Cy2, Texas Red, and AF 633-conjugated; and donkey anti-goat AF 488-conjugated antibodies. Stainings were quantified with Nikon Elements Advanced Research Image-Analysis software based on analysis of at least ten fields per section. Data is expressed as the binary area occupied by positive cells.
Flow Cytometry
Cells from tumors were pre-incubated with CD16/32 antibody (Fc block; 2.4G2; BD Pharmingen) and stained with fluorochrome-conjugated antibodies from Biolegend: BV605-CD45 (30-F11), AF700-CD3 (17A2), PerCP/Cy5.5-CD4 (GK1.5), and PE/Cy7-CD8α (53-6.7) as recommended by the manufacturer. To quantify IFN-γ expression, cells were stained with surface markers and permeabilized with Cytofix/Cytoperm (554714, BD Biosciences), and washed with 1× Perm/Wash buffer (554714, BD Biosciences) followed by incubation with PE-IFN-γ (XMG1.2, Biolegend). Prior to intracellular staining, cells were incubated in the presence of brefeldin-A and monensin (BD Biosciences) in the presence of CD3 and CD28 antibodies (17A2 and 37.51, eBioscience) adsorbed to the 96-well plates for 6 to 8 h. Data were acquired on BD Fortessa and analyzed with FlowJo software (Tree Star).
RNA was extracted from frozen tissue using RNeasy plus mini kit (Qiagen) and cDNA was generated using High-Capacity RNA-to-cDNA™ kit (Applied Biosystems). The q(RT)-PCR was performed using High Capacity cDNA Synthesis Kit, Fast SybrGreen, StepOnePlus Applied Biosystems. Relative expression was calculated using the 2−ΔΔCt method and RT2 profiler PCR Array Data Analysis (SAB Biosciences) and normalized to GAPDH. The primer sequences are as follows:
ELISA of Human and Mouse Plasma
Mouse C5a ELISA was performed according to the manufacturer's instructions (DY2150, R&D). Human complement ELISA kits C1q (HK356-02), C3 (HK366-02), C5 (HK390-02), factor B, (FB, HK367-02) factor H (FH, HK342-02), factor D (FD, HK-343-02), factor I (FI, HK355-02), C3c (HK-368). sCD59 (HK374-02), and soluble (s)C5b-9, known as membrane attack complex (MAC) or complement terminal complex (TCC) (HK328-01) were obtained from Hycult Biotech (Uden, Netherlands) and were used according to the manufacturer's recommendations.
Statistics
Data were analyzed with t-test or One-way ANOVA (more than two mean values comparison). Impact of treatments on growth of mouse tumors over time was evaluated by Two-way ANOVA. The log-rank test was used for patient survival analysis and survival data were visualized by the Kaplan-Meier estimator (The Human Protein Atlas). To determine the predictive value of complement proteins for the response to ICI the inventors used the time to next treatment (TNT) as a surrogate of response. The inventors divided patients into cohorts with protein concentration above or below a set threshold. Then, the inventors compared the distribution of TNT and calculated the p-value of a difference between the cohorts. The optimum threshold was set at the minimum p-value of separation, corresponding to a maximal difference in TNT. Multiple-protein analysis used the scikit-learn machine learning library version 0.22 [21]. The p<0.05 were considered significant. Bar graphs indicate mean±SEM. GraphPad Prism 6 was used for analyses.
High expression of complement genes is associated with unfavorable prognosis in RCC.
Using data available through the Human Protein Atlas (https://www.proteinatlas.org/) and the Cancer Genome Atlas (https://cancergenome.nih.gov/) the inventors analyzed expression of complement genes in human solid tumors (Table I). The inventors found eleven soluble complement proteins, receptors, and regulators that were associated with poor prognosis in RCC (Table I and
Complement proteins are expressed or deposited in tumor stroma and their abundance correlate with histological grade
Tumor-promoting functions of complement proteins are linked to the immunosuppressive tumor microenvironment (TME) [23]. However, some studies demonstrated that complement promotes tumor growth via direct autocrine effect on tumor cells that is independent from inhibiting antitumor T cells [24]. Therefore, it is important to determine localization of complement proteins in tumors. The inventors focused the analyses on C1qA (n=22), C1qB (n=11), C3 (n=29), and C5aR1 (n=22) because of the prognostic value of these proteins in RCC, their strategic positions in the complement cascade, and their roles in regulating tumor growth in mouse models [6]. Immunohistochemistry slides (84 samples) from 43 RCC patients, available through the Human Protein Atlas, were analyzed. C1qA was present as extracellular deposits in stroma (
Complement genes are enriched in an aggressive inflammatory subtype of RCC
The inventors previously reported the discovery of an inflammatory subtype (IS) of RCC characterized by local immune cell infiltration, systemic inflammation, poor prognosis, and BAP1 mutations [15]. In that report, the inventors used publicly available RNA-sequencing data sets from TCGA (n=529 ccRCC) as well as from UTSW KCP (n=181, including 39%, 24%, 15%, and 22% patients with ccRCC, pRCC, chromophobe, and other tumor-types, respectively). The IS cluster was enriched for gene signatures of T regulatory cells (Tregs), natural killer (NK) cells, Th1 cells, neutrophils, macrophages, B cells, CD8+ T cells, and C1q [15]. The identification of CD8+ T cells (tumor infiltrating lymphocytes-TIL) in this cluster was not surprising as CD8+ T cells have been previously associated with poor prognosis in RCC, unlike in other tumor types [25].
The inventors reanalyzed the UTSW KCP data for complement-related genes. CFB, C5AR1, CFH, C3, CIR, CIS C1QA, and C1QB were enriched in the IS, in comparison with the non-IS subtype (NIS), especially for ccRCC patients (
The direct comparison of complement genes in IS vs. NIS demonstrated higher expression of genes associated with poor prognosis in IS. In contrast, CD59, which is associated with favorable prognosis, had high expression in NIS together with another complement regulator CD46 (
Poor prognosis associated with TIL in RCC and high number of TIL in IS suggests that these T cells are dysfunctional. The inventors evaluated genes associated with T cell exhaustion/dysfunction [28] in IS vs. NIS and found that they upregulated in IS (
Complement in Plasma and the Response to Immune Checkpoint Inhibitors
In general, the contribution of non-PD-1/CTLA-4 pathways to tumor immunosuppression predicts lack of or limited response to ICI [31]. Because the high expression of complement genes is associated with: 1) T cell exhaustion, and 2) activated macrophage markers, the inventors hypothesized that complement may contribute to these immunosuppressive pathways and that increased complement activity may predict resistance to ICI. Several complement proteins are secreted from cells and circulate in plasma. Complement function is routinely evaluated in plasma in the clinic [32]. Furthermore, the inventors previously showed in mouse models that activation of complement in tumors or in metastatic sites is reflected by changes in concentrations of complement effectors in plasma [8]. Thus, the inventors evaluated complement function in plasma and sought to establish whether plasma levels of complement proteins was associated with response to ICI.
The inventors measured the concentrations of C1q, C3, C5, FB, FD, FH, FI, C3c, sCD59, and s5b-9 (TCC) in plasma collected from 24 RCC patients treated at the UTSW KCP prior to initiation of ICI (nivolumab monotherapy (n=19) or combination ipilimumab and nivolumab (n=5)) (Table 2) and from healthy donors (Oklahoma Blood Institute). The concentration of plasma complement proteins were correlated with time to next treatment (TNT), a surrogate of response to ICI. TNT is defined as a time from starting of ICI until the next line of therapy, which is usually administrated because of disease progression. The inventors used TNT as it captures patient benefit from ICI beyond the treatment course, and it is less subjective than retrospective interpretation of imaging studies, which is also influenced by the criteria used and pseudoprogression. Thus, a shorter TNT indicates a limited response to therapy. For measurements of complement proteins, the inventors used commercially available ELISA kits that are designed to work with plasma collected with different anticoagulants. However, as with other laboratory assays, the sample collection method may impact results. Therefore, the inventors sought to determine data distribution in samples collected with EDTA vs. heparin (CPT) because the analysis includes plasma collected with both anticoagulants. For all measured complement fragments, except C1q, data distributions were similar (
The inventors found that the concentration of several complement proteins in RCC patients' plasma was significantly higher than in control plasma from healthy donors except for FB. (
Next, the inventors sought to determine if using plasma concentrations of several complement proteins simultaneously would provide better prediction of response to ICI than the concentration of any single protein. To identify which combination of complement protein concentrations will be the best predictor, the inventors used the scikit-learn machine learning library [21] (http://scikit-learn.org), to establish an optimal decision tree (
Complement inhibition reduces growth of anti-PD-1-resistant renal tumors in mice by improving TIL function and inhibits angiogenesis
To test in vivo the role of complement proteins, the inventors used RCC-Renca (ATCC® CRL-2947™), a murine RCC model that while differing from human RCC, has been extensively evaluated in immunotherapy studies and is known to be recalcitrant to ICI [34], and therefore, potentially resembles ICI-resistant RCC. To evaluate the suitability of this model to study the role of complement, the inventors stained mouse tumors for complement proteins. C3 fragments were found in the vicinity of vasculature (
As previously reported [34], PD-1 blockade was ineffective in this model (
For several decades, complement was thought to contribute to cancer immune surveillance through complement-mediated lysis of tumor cells [1]. In contrast, the inventors found that complement promotes tumor growth through the inhibition of antitumor immunity, mediated via activation and recruitment of MDSC to tumors [4]. Follow-up studies documented a critical role of complement in immunosuppression in several mouse models and discovered other mechanisms involved in this process [6]. In addition to regulating TME, complement proteins and receptors directly impact tumor cells [24]. The most recent studies established a link between complement and cancer metastasis [8, 9] and demonstrated synergism between PD-1 blockade and complement inhibition [41]. Thus, there is substantial evidence from preclinical studies pointing to complement as a potential therapeutic target in cancer. Furthermore, studies using human samples indicate that early complement components (C1q, C2, and C4) are prognostic biomarkers in lung and kidney cancer [13, 42]. However, there is limited understanding of complement in human malignancies and in vivo RCC models.
The inventors found that high expression of eleven complement genes was associated with unfavorable prognosis in RCC. In contrast to recent studies [13], the inventors' analysis of large data sets from TCGA and the Human Protein Atlas failed to demonstrate a prognostic role for C4 in RCC, underscoring the need for this comprehensive analysis. No other solid tumor evaluated had such striking correlation between complement gene expression and prognosis. Supporting the same notion, the inventors found that the MAC/TCC inhibitor CD59, which inhibits complement, was associated with good prognosis. These data also indicate that limiting the final stage of complement activation may be beneficial for RCC patients. In contrast to CD59, expression of another complement regulatory protein, CD55, was not associated with improved prognosis (https://www.proteinatlas.org/ENSG00000196352-CD55/pathology). In contrast to RCC, high expression of complement genes was associated with favorable prognosis in liver, breast, pancreatic, and cervical carcinomas. Therefore, inhibiting complement may not be universally beneficial for all cancer patients and RCC seems to be an optimal target for complement-based therapy. Immunohistochemistry data confirmed the presence of complement proteins in RCC tumors. However, it is unclear where else these proteins are produced. For example, they may be synthesized in the liver and deposited in the tumor stroma and vasculature.
Data from the UTSW KCP found an association between complement gene expression and an aggressive IS of RCC, which is consistent with key roles of several complement fragments in inflammation [22]. The correlation between complement and markers of T cell exhaustion/dysfunction and alternatively activated macrophages implicates complement in regulating immunosuppression in human RCC. This is supported by the fact that the inventors found reduced expression of genes associated with T cell exhaustion as a result of complement-deficiency/inhibition in a mouse model. The contribution of C3aR1 to T cell dysfunction is further corroborated by studies of T cell-depletion showing that intact CD8+ T cells are required to reduce tumor growth in C3aR1KO mice (i.e. C3aR1 loss improves T cell function). Based on these data, complement appears to act as an additional checkpoint in RCC, which corresponds to studies indicating roles of C3aR1 and C5aR1 in the regulation of cytolytic activity of TIL in other mouse models [37]. Therefore, it is conceivable that in the presence of complement-imposed immunosuppression, therapeutic inhibition of the PD-1/CTLA-4 pathways will be ineffective. The resistance to ICI in patients with high TCC levels and low C5 levels suggest that complement activation, and possibly, subsequent complement consumption, plays a role in RCC pathogenesis. This consumption is best characterized in autoimmune diseases such as systemic lupus erythematosus and urticarial vasculitis, in which hypocomplementemia supports the diagnosis and is used to monitor disease activity [43]. Because the C1q gene signature is strongly associated with poor prognosis and aggressive IS of RCC and C1q is deposited in human and mouse tumors, the contribution of the classical pathway of complement activation to RCC should be considered. C1q colocalized with IgM in mouse tumors, therefore, the inventors theorize that these IgM may trigger the classical pathway. These IgMs may represent poly-reactive natural antibodies constitutively present in high quantities in the body fluids that bind endogenous antigens in dying, damaged, or otherwise stressed cells [44, 45]. Of note, IgM decorated apoptotic cell marked by Annexin V in mouse RCC model.
In conclusion, complement is involved in RCC pathogenesis and impacts antitumor immunity, which is reflected by its association with inflammation and poor prognosis. Targeting complement is a therapeutic option for RCC patients.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method/process steps or limitation(s)) only.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.
For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
This application claims priority to U.S. Provisional Application Ser. No. 63/084,644, filed Sep. 29, 2020, the entire contents of which are incorporated herein by reference.
This invention was made with government support under R01CA190209 and P50CA196516 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/052648 | 9/29/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63084644 | Sep 2020 | US |
