Field of invention

The present invention relates to methods for prognosis and diagnosis of breast cancer based on the activities of cellular acid-base transporters.

Background of invention

Transport of acid-base equivalents across cell membranes forms the foundation for intracellular pH control in normal and cancer tissue. Due to the high metabolic rate and consequent increased production of acidic waste, cancer cells are very dependent on net acid extrusion that defend against intracellular acidification and thereby maintain intracellular pH in a range permissive for cell metabolism, survival, and proliferation. Breast cancer is a heterogeneous disease with inter-individual variation, for instance, in histopathology and expression of growth factor and sex hormone receptors. These differences in pro-oncogenic signaling have consequences for prognosis and treatment responses in patients with breast cancer. In general, net acid extrusion in breast cancer tissue takes place via Na ⁺ ,HC0 ₃ ^" -cotransport and Na7H ⁺ -exchange with potential additional contribution from H ⁺ -ATPases and monocarboxylate transporters. However, it has not been resolved to what extent the relative contribution of these distinct acid- base transport mechanisms is uniform or varies between patients with breast cancer.

Acid-base transporters are potential targets for anti-cancer therapy and knowledge regarding predominant acid-base transport functions in cancer tissue from individual patients would provide vital information for predicting treatment responses to inhibitors of acid-base transporters. Acid-base transporters and local pH modify cell proliferation, migration, and extracellular matrix degradation, and variability in acid-base transport mechanisms in normal breast tissue and malignantly transformed breast tissue may therefore contribute to differences in risk for breast cancer development and the potential for and rate of cancer progression including the risk of metastatic

dissemination. Being able to accurately predict treatment responses and the risk of disease development and dissemination would greatly improve the possibilities for rationally designing screening and treatment protocols with superior treatment outcome and reduced risk of side-effects. Summary of invention

A main object of the present invention is to provide prognostic and diagnostic methods, which are based on measurements of the acid-base transport across the cellular membrane. Regulation of acid-base transport is import to maintain intracellular pH, and changes in acid-base transport are important for cancer development.

In one aspect, the present invention relates to a method for determining the prognosis of a breast cancer, said method comprising

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na ⁺ /H ⁺ exchange activity in cells of a sample from a patient,

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation, and

c) selecting a patient for whom the relative importance of Na7HC0 ₃ ^" co- transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value.

In another aspect, a method is provided of identifying a patient with increased risk of developing metastatic breast cancer, said method comprising

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from said patient, and

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation, and

c) comparing the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation with a predetermined value.

In another aspect, a method is provided of selecting a breast cancer patient for treatment with an inhibitor of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange, said method comprising

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from a patient, and

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or

Na7H ⁺ exchange for intracellular pH regulation, and c) selecting patients for whom the relative importance of Na7HC0 ₃ ^" co- transport and/or Na ⁺ /H ⁺ exchange for intracellular pH regulation is above or below a predetermined value.

In yet another aspect, a method is provided of treating breast cancer, said method comprising

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na ⁺ /H ⁺ exchange activity in cells of a sample from a patient,

b) selecting a patient for whom the relative importance of Na7HC0 ₃ ^" co- transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value, and

c) providing an inhibitor of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange to said patient.

One aspect also relates to a use of an inhibitor of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for treating a breast cancer in a patient, wherein the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value. Description of Drawings

Figure 1. The capacity for pH recovery from intracellular acidification is increased in human breast cancer tissue compared to normal breast tissue both in the presence and absence of C0 ₂ /HC0 ₃ ^" . A. Average traces of intracellular pH (pH,) during NH ₄ ⁺ - prepulse experiments performed on organoids isolated from human breast cancer and normal breast tissue (n=27-28). B. Rates of Na ⁺ -dependent intracellular pH (pH,) recovery plotted as function of intracellular pH in organoids from breast cancer and normal breast tissue (n=27-28). The displayed lines are the result of least-squares linear regression analyses and are all significantly different (P<0.01 or P<0.001 ). C. Steady-state intracellular pH (pH,) in organoids isolated from breast cancer and normal breast tissue (n=27-28). Intracellular pH is elevated in cancer tissue compared to normal breast tissue (P<0.001 ; two-way ANOVA) and higher in the presence than absence of C0 ₂ /HC0 ₃ ^" (P<0.001 ; two-way ANOVA). Figure 2. Na ⁺ ,HC0 ₃ ^~ -cotransport and C0 ₂ /HC0 ₃ ^~ -dependent changes in steady-state intracellular pH are accentuated in HER2-positive breast cancer tissue. A+B. Average traces of intracellular pH (pH,) during NH ₄ ⁺ -prepulse experiments performed on organoids isolated from human breast cancer (A) and normal breast (B) tissue (n=3- 25). The data set presented in Figure 1 is here divided according to HER2 receptor status. C+D. Rates of Na ⁺ -dependent intracellular pH (pH,) recovery plotted as function of intracellular pH in organoids from breast cancer (C) and normal breast (D) tissue (n=3-25). The data set presented in Figure 1 is here divided according to HER2 receptor status. The displayed lines are the result of least-squares linear regression analyses and the stars indicate levels of significance ( ^* P<0.05, ^*** P<0.001 ) when tissue from women with HER2-positive breast cancer is compared to the same type of tissue (cancer or normal) from women with HER2-negative breast cancer. E. Steady- state intracellular pH (pH,) in organoids isolated from human breast cancer and normal breast tissue (n=3-25). The data set presented in Figure 1 is here divided according to HER2 receptor status. The effect of removing C0 ₂ /HC0 ₃ ^" on steady-state intracellular pH is greater in HER2-positive than HER2-negative breast cancer tissue (interaction: P<0.01 , two-way ANOVA) but not significantly different between normal breast tissue from women with HER2-positive and HER2-negative breast cancer (interaction:

P=0.98, two-way ANOVA).

Figure 3. Na ⁺ ,HC0 ₃ ^~ -cotransport and C0 ₂ /HC0 ₃ ^" -dependent changes in steady-state intracellular pH are attenuated in normal breast tissue and breast cancer tissue from women with lymph node metastases. A+B. Average traces of intracellular pH (pH,) during NH ₄ ⁺ -prepulse experiments performed on organoids isolated from human breast cancer (A) and normal breast (B) tissue (n=6-22). The data set presented in Figure 1 is here divided according to whether the women were found to have lymph node metastases (including micro-metastases and individual tumor cell metastases) or not. C+D. Rates of Na ⁺ -dependent intracellular pH (pH,) recovery plotted as function of intracellular pH in organoids from breast cancer (C) and normal breast (D) tissue (n=6- 22). The data set presented in Figure 1 is here divided according to whether the women were found to have lymph node metastases (including micro-metastases and individual tumor cell metastases) or not. The displayed lines are the result of least- squares linear regression analyses and the stars indicate levels of significance

( ^* P<0.05, ^*** P<0.001 , NS: not significantly different) when tissue from women with lymph node metastases is compared to the same type of tissue (cancer or normal) from women without lymph node metastases. E. Steady-state intracellular pH (pH,) in organoids isolated from human breast cancer and normal breast tissue (n=6-22). The data set presented in Figure 1 is here divided according to whether the women were found to have lymph node metastases (including micro-metastases and individual tumor cell metastases) or not. There is a strong tendency (P=0.06, two-way ANOVA) for a greater effect of removing C0 ₂ /HC0 ₃ ^" on steady-state intracellular pH in breast cancer and normal breast tissue from women with identified lymph node metastases compared to women without lymph node metastases. Figure 4. The relative contribution of Na ⁺ ,HC0 ₃ ^" -cotransport and Na7H ⁺ -exchange to intracellular pH control differs between breast cancer of different histopathologies. A+D. Average traces of intracellular pH (pH,) during NH ₄ ⁺ -prepulse experiments performed on organoids isolated from human breast cancer and normal breast tissue. The data set presented in Figure 1 is here divided according to histopathology between patients with invasive ductal carcinoma (A, n=18) and invasive lobular carcinoma (D, n=5). Less frequently occurring histopathologies are not displayed. B+E. Rates of Independent intracellular pH (pH,) recovery plotted as function of intracellular pH in organoids from breast cancer and normal breast tissue divided according to

histopathology of the primary breast tumor between patients with invasive ductal carcinoma (B, n=18) and invasive lobular carcinoma (E, n=5). The displayed lines are the result of least-squares linear regression analyses; and the stars indicate levels of significance ( ^*** P<0.001 , NS: not significantly different) when organoids examined in the presence of C0 ₂ /HC0 ₃ ^" are compared with organoids isolated from the same type of tissue investigated in the absence of C0 ₂ /HC0 ₃ ^" . C+F. Steady-state intracellular pH (pHj) in organoids isolated from breast cancer and normal breast tissue sampled from women with invasive ductal carcinoma (C) and invasive lobular carcinoma (F).

Intracellular pH is elevated in cancer tissue compared to normal breast tissue (P<0.01 for invasive ductal carcinoma, P<0.05 for invasive lobular carcinoma; two-way ANOVA) and higher in the presence than absence of C0 ₂ /HC0 ₃ ^" (P<0.01 for both

histopathologies; two-way ANOVA).

Figure 5. Steady-state intracellular pH in breast cancer tissue is not dependent on the size of the primary breast tumor. The data set presented in Figure 1 C is here plotted as function of primary tumor size. The lines are the result of least-squares linear regression analyses; the slopes are not significantly different from 0 (P=0.14 with C0 ₂ /HC0 ₃ ^" , P=0.92 without C0 ₂ /HC0 ₃ ^" ).

Figure 6. Intracellular intrinsic buffering capacity was not significantly different between organoids isolated from breast cancer tissue and corresponding normal breast tissue. The intrinsic buffering capacity was calculated from experiments performed in absence of CO2/HCO ₃ ^" . Values were log-transformed and slopes and intercepts from least- squares linear regression analyses were not significantly different when organoids isolated from breast cancer tissue were compared with organoids from normal breast tissue (slopes: P=0.57, intercepts: P=0.23). The displayed line provides the best-fit linear representation of the pooled data set.

Figure 7. Breast cancer tissue has increased capacity for net acid extrusion and elevated intracellular pH compared to normal breast tissue. A. Average traces (n=49- 51 ) of intracellular pH during NH ₄ ⁺ prepulse experiments; organoids isolated from normal breast tissue and breast cancer tissue were investigated in presence and absence of C0 ₂ /HC0 ₃ ^~ . B. Na ⁺ -dependent net base uptake (equal to net acid extrusion) grouped in normal and cancer tissue investigated in presence and absence of CO2/HCO ₃ ^" (n=49-51 ). Each symbol represents the mean transport activity with SEM plotted as function of the corresponding average intracellular pH. ^*** P<0.001 , NS: not significantly different vs. C0 ₂ /HC0 ₃ ^" -free conditions in tissue of the same source (normal breast or breast cancer) when compared by least-squares linear regression analysis. C. Steady-state intracellular pH in organoids isolated from breast cancer and normal breast tissue and investigated in presence or absence of C0 ₂ /HC0 ₃ ^" (n=49-51 ). Intracellular pH is elevated in cancer tissue compared to normal tissue in presence ( ^*** P<0.001 ) and absence ( ^*** P<0.001 ) of C0 ₂ /HC0 ₃ ^" ; data were compared by two- way ANOVA followed by Bonferroni multiple comparison tests.

Figure 8. Na ⁺ ,HC0 ₃ ^" -cotransport and C0 ₂ /HC0 ₃ ^" -dependent changes in steady-state intracellular pH are increased in HER2-positive (HER2 ⁺ ) compared to HER2-negative (HER2 ^" ) breast cancer tissue. A+B. Average traces of intracellular pH during NH ₄ ⁺ prepulse experiments performed on normal and cancer organoids from women with HER2-positive (A, n=7) or HER2-negative (B, n=42) breast tumors. C. Steady-state intracellular pH in organoids from breast cancer tissue and normal breast tissue divided according to HER2-status. The effect of removing C0 ₂ /HC0 ₃ ^" on steady-state intracellular pH is greater in HER2-positive compared to HER2-negative cancer tissue (interaction: ^* P<0.05; two-way ANOVA). This effect is not observed in the normal tissue (P=0.42; two-way ANOVA). D+E. Na ⁺ -dependent net base uptake as function of intracellular pH in HER2-postive (D) and HER2-negative (E) breast tissue. Transport activities were analyzed by least-squares linear regression analyses. ^* P<0.05, ^*** P<0.001 , NS: not significantly different vs. C0 ₂ /HC0 ₃ ^" -free conditions.

Figure 9. Na ⁺ ,HC0 ₃ ^~ -cotransport is increased in estrogen receptor negative (0-10% ER ⁺ ) compared to estrogen receptor positive (90-100% ER ⁺ ) breast cancer tissue. A+B. Average traces of intracellular pH during NH ₄ ⁺ prepulse experiments performed on normal and cancer organoids from women with downregulation of estrogen receptors (0-10% ER ⁺ , A; n=5) or normal estrogen receptor status (90-100% ER ⁺ , B; n=44) in their primary tumor. C. Steady-state intracellular pH in organoids isolated from breast cancer tissue and normal breast tissue divided according to ER status. Estrogen receptor downregulation did not affect the C0 ₂ /HC0 ₃ ^" -dependent changes in steady- state intracellular pH in cancer or normal tissue (interaction; P=0.75 for cancer and P=0.47 for normal; two-way ANOVA). D+E. Na ⁺ -dependent net base uptake as function of intracellular pH in estrogen receptor negative (D) and estrogen receptor positive (E) tissue. Transport activities were analyzed by least-squares linear regression analyses. ^** P<0.01 , ^*** P<0.001 , NS: not significantly different vs. C0 ₂ /HC0 ₃ ^" -free conditions.

Figure 10. The C0 ₂ /HC0 ₃ ^" -dependency of net base uptake and steady-state intracellular pH is attenuated in primary tumors from women with lymph node (LN) metastases. A+B. Average traces of intracellular pH during NH ₄ ⁺ prepulse experiments performed on organoids isolated from normal and breast cancer tissue from women with macro-, micro-, or single tumor cell metastases to the regional lymph nodes (A, n=13) and women without metastases (B, n=35). C. Steady-state intracellular pH in organoids isolated from breast cancer tissue and normal breast tissue grouped according to regional lymph node status. There is a strong tendency (P=0.05, two-way ANOVA) for a greater effect of removing C0 ₂ /HC0 ₃ ^" in primary breast cancer tissue from women with no lymph node metastases compared to women with lymph node metastases. D+E. Na ⁺ -dependent net base uptake as function of intracellular pH in breast tissue from women with (D) or without (E) regional lymph node metastases. Transport activities were analyzed by least-squares linear regression analyses.

*P<0.05, NS: not significant different vs. C0 ₂ /HC0 ₃ ^" -free conditions. Figure 11. Na ⁺ ,HC0 ₃ ^~ -cotransport and C0 ₂ /HC0 ₃ ^~ -dependent changes in steady-state intracellular pH are increased in invasive lobular carcinomas (ILC) compared to invasive ductal carcinomas (IDC). A+B. Average traces of intracellular pH during NH ₄ ⁺ prepulse experiments performed on organoids isolated from breast cancer tissue and normal breast tissue from women with IDC (A, n=39) or ILC (B, n=5). The remaining histopathologies are not displayed because of low sample numbers and consequent low statistical power in each group. C. Steady-state intracellular pH in organoids from breast cancer tissue and normal breast tissue divided according to histopathology. The effect of removing C0 ₂ /HC0 ₃ ^" on steady-state intracellular pH is greater in cancer tissue from women with ILC compared to IDC (interaction ^* P<0.05; two-way ANOVA). This effect is not observed in normal tissue (interaction P=0.54; two-way ANOVA). D+E. Na ⁺ -dependent net base uptake as function of intracellular pH in tissue from patients with IDC (D) or ILC (E). Transport activities were analyzed by least-squares linear regression analyses. ^*** P<0.001 , NS: not significantly different vs. C0 ₂ /HC0 ₃ ^~ - free conditions.

Figure 12. Steady-state intracellular pH in organoids isolated from breast cancer tissue and normal breast tissue is not dependent on primary tumor size or the age of the patient at diagnosis. A. Steady-state intracellular pH plotted as function of the log- transformed tumor size (n=49-51 ). Lines are the results of least-squares linear regression analyses; the slopes are not significantly different from 0 (P=0.76 and P=0.93 for normal breast tissue in presence and absence of C0 ₂ /HC0 ₃ ^" , respectively; P=0.54 and P=0.76 for breast cancer tissue in presence and absence of C0 ₂ /HC0 ₃ ^~ respectively. B Steady-state intracellular pH plotted as function of patient age at time of breast cancer diagnosis. Lines are the results of least-squares linear regression analyses; the slopes are not significantly different from 0 (P=0.92 and P=0.19 for normal tissue in presence and absence of C0 ₂ /HC0 ₃ ^~ respectively; P=0.49 and P=0.67 for cancer tissue in presence and absence of C0 ₂ /HC0 ₃ ^~ respectively.

Detailed description of the invention

Prognostic and diagnostic methods are provided herein, which are based on measurements of the acid-base transport across the cellular membrane. Acid-base transport is vital for intracellular pH regulation, and it is particularly important in cancer tissue due to the high metabolic rate and consequent increased production of acidic waste. Thus cancer cells are very dependent on net acid extrusion that defend against intracellular acidification and thereby maintain intracellular pH in a range permissive for cell metabolism, survival, and proliferation.

It has surprisingly been found that the mechanisms of intracellular pH regulation— i.e., the C0 ₂ /HC0 ₃ ^" -dependency of net acid extrusion and steady-state intracellular pH— varies substantially between groups of patients. The invention therefore relates to methods for determining the prognosis of a breast cancer, such as identifying a patient with increased risk of developing metastatic breast cancer; selecting a breast cancer patient for treatment with an inhibitor of Na7HC0 ₃ ^" co-transport and/or Na ⁺ /H ⁺ exchange; and also treating breast cancer, where the prognosis, risk, selection and treatment is based on the activity of Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from the respective patient. Thus, the invention provides a method for determining the prognosis of a breast cancer, said method comprising

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from a patient,

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺

exchange for intracellular pH regulation, and

c) selecting a patient for whom the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value. A method is also provided of identifying a patient with increased risk of developing metastatic breast cancer, said method comprising

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from said patient, and

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺

exchange for intracellular pH regulation, and

c) comparing the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺

exchange for intracellular pH regulation with a predetermined value. Moreover, a method is provided of selecting a breast cancer patient for treatment with an inhibitor of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange, said method comprising

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na ⁺ /H ⁺ exchange activity in cells of a sample from a patient, and

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation, and

c) selecting patients for whom the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value.

A method is also provided of treating breast cancer, said method comprising a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from a patient,

b) selecting a patient for whom the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value, and

c) providing an inhibitor of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange to said patient.

A use of an inhibitor of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange is also provided for treating a breast cancer in a patient, wherein the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value.

More specifically, a use is provided of

a) an inhibitor of Na7HC0 ₃ ^" co-transport for treating a breast cancer in a patient, wherein the relative importance of Na7HC0 ₃ ^" co-transport is above a predetermined value and/or

b) an inhibitor of Na7H ⁺ exchange for treating a breast cancer in a patient, wherein the relative importance of Na7H ⁺ exchange for intracellular pH regulation is above a predetermined value. In the context of the present disclosure, the term "relative importance" with respect to the cellular acid-base regulators is meant to refer to the extent to which each of the two transporters, Na7HC0 ₃ ^" co-transporters and Na ⁺ /H ⁺ exchangers, compared with each other is responsible for regulation of the intracellular pH. The relative importance of Na7HC0 ₃ ^" co-transport and/or Na ⁺ /H ⁺ exchange for intracellular pH regulation can be determined in a trace experiment as shown in figure 1. Higher contribution from Na ⁺ /H ⁺ exchange to intracellular pH regulation in a tissue sample from a patient is observed as an ability of the cells in the absence of C0 ₂ /HC0 ₃ ^" to recover intracellular pH faster after intracellular acidification and/or at more alkaline intracellular pH levels than in a sample with lower contribution from Na7H ⁺ -exchange activity. The contribution from Na7HC0 ₃ ^" co-transport to intracellular pH regulation in a tissue sample from a patient is evaluated based on the increased ability to recover intracellular pH after intracellular acidification (i.e., faster recovery and/or recovery at more alkaline intracellular pH levels) when investigated in the presence compared to the absence of C0 ₂ /HC0 ₃ ^" ; and a higher contribution from Na7HC0 ₃ ^" co-transport in a sample is observed as a greater increase in the ability to recover intracellular pH from intracellular acidification compared to a sample with lower contribution from Na7HC0 ₃ ^" co-transport.

However, it has not been resolved to what extent the relative contribution of these distinct acid-base transport mechanisms is uniform or varies between patients with breast cancer.

Acid-base transporters

The transport of acid-base equivalents across cell membranes is governed by four types of transporters, Na ⁺ ,HC0 ₃ ^" -cotransporters (NBC); Na7H ⁺ -exchangers (NHE); H ⁺ - ATPases (proton pumps) and monocarboxylate transporters (MCT). In breast cancer tissue, the net acid extrusion in breast cancer tissue takes place via Na ⁺ ,HC0 ₃ ^~ - cotransport and Na7H ⁺ -exchange, although there may be an additional contribution from H ⁺ -ATPases and monocarboxylate transporters. Thus, in the methods and uses disclosed herein, the activity of acid-base transporters is preferably determined for Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange. In particular, the Na7HC0 ₃ ^" co-transport activity is determined and/or the Na7H ⁺ exchange activity is determined. Upon determination of the respective activities of the acid-base transporters, their relative importance for the intracellular pH regulation is determined. In one preferred embodiment, the Na7HC0 ₃ ^" co-transport activity is determined and the relative importance of Na7HC0 ₃ ^" co-transport for intracellular pH regulation is determined. In another preferred embodiment, the Na ⁺ /H ⁺ exchange activity is determined and the relative importance of Na ⁺ /H ⁺ exchange for intracellular pH regulation is determined.

Na7HC0 ₃ ^" co-transport is mediated by proteins of the solute carrier 4 (SLC4) family. This family comprise at least 1 1 members and include electrogenic, electroneutral and Na ⁺ -driven co-transporters. Thus, generally, the methods and uses of the present invention may include determining Na7HC0 ₃ ^" co-transport activity mediated by any member of the SLC4 family. However, in a more preferred embodiment, the methods and uses comprises determining Na7HC0 ₃ ^" co-transport activity mediated by an electroneutral Na7HC0 ₃ ^" co-transport, such as SLC4A7 (aka NBCnl ).

In parallel Na7H ⁺ exchange is mediated by another family of solute carrier proteins known as the SLC9-family. Mammalian Na7H ⁺ exchangers of the SLC9 family are widely expressed and involved in numerous essential physiological processes. Their primary function is to mediate the 1 :1 exchange of Na ⁺ for H ⁺ across the cellular membrane. At least 1 1 members exist in this family, and generally, the methods and uses of the present invention may include determining Na7H ⁺ exchange activity mediated by any member of the SLC9 family. However, in a more preferred embodiment, the methods and uses comprises determining Na7H ⁺ exchange activity mediated by SLC9A1 (aka NHE1 ).

In addition, the methods and uses provided herein for treatment of breast cancer include providing an inhibitor of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange to the patient. In more specific embodiments, the inhibitor is an NBCnl [SLC4A7] inhibitor or the inhibitor is an NHE1 [SLC9A1] inhibitor.

Prognosis and diagnosis

One aspect of the invention relates to a method for breast cancer prognosis. The inventors have surprisingly found that there is a correlation between important prognostic markers for breast cancer and the activities of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange in a patient sample. For example, expression of human epidermal growth factor receptor 2 (HER2, a.k.a. ErbB2), presence of lymph node metastases, and histopathology shows specific activity profiles of acid-base transporters.

Thus, a prognostic method is provided for the prognosis of a breast cancer, said method comprising

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na ⁺ /H ⁺ exchange activity in cells of a sample from a patient,

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or Na ⁺ /H ⁺ exchange for intracellular pH regulation, and

c) selecting a patient for whom the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value.

In another aspect, the method comprises

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from a patient,

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation, and

c) determining the prognosis based on the relative importance of Na7HC0 ₃ ^" co- transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value.

Alternatively, step c) in either method above may comprise identifying a patient for whom the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value as a patient with a poor prognosis.

These methods can be used to infer important prognostic markers. Specifically, the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is in preferred embodiments indicative of HER2 receptor overexpression or gene amplification, lymph node metastasis, tumour type, tumour size, estrogen receptor status or malignancy grade. In another aspect, a diagnostic method is provided for breast cancer, which is based on the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation. In one aspect, a method is provided for identifying a patient with increased risk of developing metastatic breast cancer, said method comprising a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from said patient, and

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or Na+/H+ exchange for intracellular pH regulation, and

c) comparing the relative importance of Na7HC0 ₃ ^" co-transport and/or Na+/H+ exchange for intracellular pH regulation with a predetermined value.

More specifically, the diagnostic method for determining the presence, risk and/or prognosis of breast cancer in a patient may comprise the steps of:

a) providing or obtaining a sample, such as a cancer tissue sample, from said patient

b) determining the Na+/ HC0 ₃ ^" co-transport activity in cells of said sample, c) determining the Na+/H+ exchange activity in cells of a sample, a) establishing the relative importance of Na+/ HC0 ₃ ^" co-transport and/or

Na+/H+ exchange for intracellular pH regulation, and

b) comparing the relative importance of Na+/ HC0 ₃ ^" co-transport and/or

Na+/H+ exchange for intracellular pH regulation with a predetermined value, c) determining the presence, risk and/or prognosis of breast cancer in said patient on the basis of the comparison of the relative importance of Na+/ HC0 ₃ ^" co- transport and/or Na+/H+ exchange for intracellular pH regulation with a predetermined value.

A method is also provided for selecting a breast cancer patient for treatment with an inhibitor of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange, said method comprising a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from a patient, and

b) determining the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺

exchange for intracellular pH regulation, and

c) selecting patients for whom the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value. More specifically, the method for selecting a breast cancer may comprise the steps of: a) providing or obtaining a sample, such as a cancer tissue sample, from said patient

b) determining the Na+/ HC0 ₃ ^" co-transport activity in cells of said sample, c) determining the Na+/H+ exchange activity in cells of a sample, d) establishing the relative importance of Na+/ HC0 ₃ ^" co-transport and/or Na+/H+ exchange for intracellular pH regulation, and

e) comparing the relative importance of Na+/ HC0 ₃ ^" co-transport and/or

Na+/H+ exchange for intracellular pH regulation with a predetermined value, f) selecting a breast cancer patient for treatment with an inhibitor of

Na7HC0 ₃ ^" co-transport and/or Na ⁺ /H ⁺ exchange on the basis of the comparison of the relative importance of Na+/ HC0 ₃ ^" co-transport and/or Na+/H+ exchange for intracellular pH regulation with a predetermined value.

In this approach, the choice of therapy is determined on the basis of the relative importance of Na7HC0 ₃ ^" co-transport and/or Na ⁺ /H ⁺ exchange for intracellular pH regulation.

In the methods and uses provided herein, a patient is in one embodiment selected for treatment with a modulator, such as an inhibitor, of Na7HC0 ₃ ^" co-transport when the relative importance of Na7HC0 ₃ ^" co-transport for intracellular pH regulation is above or below a predetermined value, in which case a modulator, such as an inhibitor, of Na7HC0 ₃ ^" co-transport can be provided for treatment of the breast cancer. It is preferred that patient is selected for treatment with an inhibitor of Na7HC0 ₃ ^" co- transport when the relative importance of Na7HC0 ₃ ^" co-transport for intracellular pH regulation is above a predetermined value, in which case an inhibitor of Na7HC0 ₃ ^" co- transport is provided for treatment of the breast cancer.

However, in another embodiment, a patient is selected for whom the relative importance of Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value, in which case a modulator, such as an inhibitor, of Na7H ⁺ exchange can be provided. Still, it is preferred that a patient is selected for whom the relative importance of Na7H ⁺ exchange for intracellular pH regulation is above a predetermined value, in which case an inhibitor, of Na7H ⁺ exchange can be provided.

In one embodiment of the methods or uses provided herein, the breast cancer is a triple negative breast cancer, an estrogen receptor-positive breast cancers and/or a HER2-positive breast cancer. Specifically, the methods for prognosis of a breast cancer provided herein relates to determining whether the breast cancer is a triple negative breast cancer, an estrogen receptor-positive breast cancers and/or a HER2- positive breast cancer.

Specifically, the inventors have found that breast cancer tissue from women with lymph node metastases differs in intracellular pH regulatory function from breast cancer tissue obtained from women without lymph node metastases: the contribution from

Na7HC0 ₃ ^" cotransport to net acid extrusion and intracellular steady-state pH regulation is low in the primary breast cancer tissue and normal breast tissue from women with metastatic dissemination to regional lymph nodes. The difference in intracellular pH regulation in normal breast tissue shows that variation in normal tissue function can predispose to metastatic breast cancer disease. The invention, therefore, specifically relates to a prognostic and therapeutic method, wherein a reduced relative importance of Na7HC0 ₃ ^" co-transport is indicative of increased risk of developing metastatic breast cancer. Such methods may also be used to infer a suitable treatment or monitorization program in order to prevent or ameliorate the cancer progress.

Sample

The methods and uses provided herein for diagnosis, prognosis, risk assessment or treatment generally comprises determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from a patient. The sample can be any relevant suitable biological sample comprising live cells from the patient. However, in a preferred embodiment, the sample is a tissue sample, preferably a sample of breast tissue. The tissue may be cancer derived, for example be taken from a tumour, in certain embodiments, such as methods and uses related to treatment and prognosis of breast cancer. However, the sample may also comprise or consist of normal tissue, for example taken from breast tissue adjacent to a tumour or from a woman without diagnosed cancer. The sample can be a biopsy taken by usual methods available in the art. In one embodiment, the sample is a needle biopsy such as a core needle biopsy (CNB), however a fine needle biopsy (FNAB) can also be employed. If the area to be biopsied can be felt, the medical professional locates the lump or suspicious area and guides the needle there. If the lump can't be felt, ultrasound might be used to watch the needle on a screen as it moves toward and into the mass

(ultrasound-guided biopsy). The medical professional may also use stereotactic needle biopsy to guide the needle. For a stereotactic needle biopsy, computers map the exact location of the mass using mammograms taken from 2 angles, which is then used to guide the needle to the right spot. A computer then pinpoints exactly where in the abnormal area the needle tip needs to go. Once the needle is in place, tissue is drawn out. Vacuum-assisted biopsies can be done with special biopsy systems. For these procedures, the skin is numbed and a small cut (less than ¼ inch) is made. A hollow probe is put in through the cut and guided into the abnormal area of breast tissue using x-rays, ultrasound, or MRI. Tissue is then pulled into the probe through a hole in its side, and a rotating knife inside the probe cuts the tissue sample from the rest of the breast. This allows multiple tissue samples to be removed through one small opening.

Mostly, the breast cancer diagnostic and prognostic methods provided herein are based on needle biopsies as described above. However, in some cases a surgical biopsy may be needed. A surgical biopsy is done by cutting the breast to take out all or part of the lump so it can be looked at under a microscope. In such cases of surgical removal, biopsy tissue samples can be obtained from the removed material. This may also be called an open biopsy. There are generally 2 types of surgical biopsies: 1 ) incisional biopsy removes only part of the suspicious area, enough to make a diagnosis and 2) excisional biopsy removes the entire tumor or abnormal area, with or without trying to take out an edge of normal breast tissue (it depends on the reason for the excisional biopsy).

Medical use and treatment

The present disclosure provides methods for prognosis and diagnosis of breast cancer. These methods can also be used to determine a specific suitable treatment for the relevant patients, for whom the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value. For example, breast cancer patients can be selected for a specific treatment with modulators, such as inhibitors, of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange.

In one aspect, a method is provided of treating breast cancer, the method comprising a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from a patient,

b) selecting a patient for whom the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a

predetermined value, and

c) providing a modulator, such as an inhibitor, of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange to said patient. More specifically, the method may comprise the steps of:

a) providing or obtaining a sample, such as a cancer tissue sample, from said patient

b) determining the Na+/ HC0 ₃ ^" co-transport activity in cells of said sample, c) determining the Na+/H+ exchange activity in cells of a sample, d) establishing the relative importance of Na+/ HC0 ₃ ^" co-transport and/or

Na+/H+ exchange for intracellular pH regulation, and

e) comparing the relative importance of Na+/ HC0 ₃ ^" co-transport and/or Na+/H+ exchange for intracellular pH regulation with a predetermined value, f) selecting a patient for whom the relative importance of Na7HC0 ₃ ^" co- transport or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value, and

g) providing a modulator, such as an inhibitor, of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange to said patient In this approach, the choice of therapy is determined on the basis of the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation. One aspect also provides a use of a modulator, such as an inhibitor, of Na7HC0 ₃ ^" co- transport and/or Na ⁺ /H ⁺ exchange for treating a breast cancer in a patient, wherein the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a predetermined value.

In one embodiment, the method of treating breast cancer comprises

a) determining the Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity in cells of a sample from a patient,

b) selecting a patient for whom the relative importance of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange for intracellular pH regulation is above or below a

predetermined value, and

c) providing an inhibitor, of Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange to said patient. It is preferred that patient is selected for treatment with an inhibitor of Na7HC0 ₃ ^" co- transport when the relative importance of Na7HC0 ₃ ^" co-transport for intracellular pH regulation is above a predetermined value, in which case an inhibitor of Na7HC0 ₃ ^" co- transport is provided for treatment of the breast cancer. Still, it is preferred that a patient is selected for whom the relative importance of Na7H ⁺ exchange for intracellular pH regulation is above a predetermined value, in which case an inhibitor, of Na7H ⁺ exchange can be provided.

For example, the therapeutic methods and uses comprises providing or administering an inhibitor of Na7HC0 ₃ ^" co-transport. In another embodiment, the therapeutic methods and uses comprises providing or administering an inhibitor of Na7H ⁺ exchange.

In one preferred embodiment, the inhibitor is an inhibitor of Na7HC0 ₃ ^" co-transport; i.e. an inhibitor of a gene product of the SLC4-family, preferably an inhibitor of SLC4A7 (NBCnl ).

The inhibitor may be any relevant compound, such as an antibody or a small molecule. In one embodiment, the small molecule is cariporide. Cariporide is a selective inhibitor of Na7H ⁺ exchange, and thus in one embodiment, Cariporide is provided for use in treatment of breast cancer in a patient, wherein the relative importance of Na ⁺ /H ⁺ exchange for intracellular pH regulation is above a predetermined value.

However, the inhibitor may also be an antibody or antigen binding fragment thereof targeting a gene product of the SLC4-family and/or the SLC9-family, in particular SLC4A7 (NBCnl ) or SLC9A1 (NHE1 ). In a preferred embodiment, the antibody or antigen binding fragment thereof specifically recognize and bind an extracellular polypeptide region of gene product of the SLC4-family and/or the SLC9-family, in particular SLC4A7 (NBCnl ) or SLC9A1 (NHE1 ). More preferred, the antibody or antigen binding fragment thereof specifically recognize and bind an extracellular polypeptide region comprises or consists of a sequence selected from any one of SEQ ID NO: 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 or a fragment thereof. Thus, the extracellular polypeptide region may comprise or consist of a sequence selected from SEQ ID NO: 14, 15 and 16. Obviously, the antibody or antigen binding fragment thereof is preferably capable of inhibiting the cellular function of the SLC4 protein family, such as NBCnl -mediated Na ⁺ -dependent HC0 ₃ ^" transport and/or SLC9 protein family, such as NHE1 -mediated Na ⁺ -dependent hTtransport.

The therapeutic methods and uses provided herein may further comprise providing a further therapeutic agent, such as an additional anti breast cancer agent. For example, an estrogen-receptor modulator or a HER2 antibody could be provided or administered, or a regulator of an ion transporter, such as a Na7H ⁺ exchanger (NHE), a

monocarboxylic acid transporter (MCT) or a proton pump inhibitor (PPI) could be provided. The treatment can be applied at any stage of a breast cancer and can also be used for prophylactic treatment of breast cancer. Thus, the treatment according to the present invention involves both prophylactic and curative treatment as well as prevention and amelioration of symptoms or associated with a breast cancer and prevention of disease progression.

The therapeutic methods and uses could be combined with chemotherapy and/or radiotherapy. The uses, medicaments and methods of treatment may also be used before or after surgical therapy. Antibody or antigen binding fragment

An antibody or antigen binding fragment thereof is provided for use in treatment of breast cancer, which antibody is capable of targeting and modulating the activity of a gene product of the SLC4-family and/or the SLC9-family, in particular SLC4A7

(NBCnl ) or SLC9A1 (NHE1 ). In a preferred embodiment, the provided antibody is capable of inhibiting either Na7HC0 ₃ ^" co-transport activity and/or Na7H ⁺ exchange activity.

In a preferred embodiment, the antibody is capable of inhibiting cellular NBCnl - mediated Na ⁺ -dependent HC0 ₃ ^" transport. The antibody or antigen binding fragment thereof preferably specifically recognizes and binds an extracellular polypeptide region of human NBCnl . In a preferred embodiment, the extracellular polypeptide region comprises or consists of a sequence selected from any one of SEQ ID NO: 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 or a fragment thereof. In one particular embodiment, the extracellular polypeptide region comprises or consists of a human sequence selected from any one of SEQ ID NO: 14, 15, 16 or a fragment thereof, such as a fragment of 5- 25, such as 10-25, such as 10-20 amino acids thereof. In one embodiment, the antibody or antigen binding fragment thereof is capable of inhibiting cellular NBCnl - mediated Na ⁺ -dependent HC0 ₃ ^" transport.

The antibody may be any polypeptide or protein capable of recognising and binding an antigen in a gene product of the SLC4 or SLC9 families, in particular NBCnl or NHE1 , preferably an extracellular polypeptide region. Preferably, the antibody is capable of specifically binding said antigen. By the term "specifically binding" is meant binding with at least 10 times higher affinity to the antigen than to a non-specific antigen (e.g. BSA). Typically, the antibody binds with an affinity corresponding to a K _D of about 10 ^"7 M or less, such as about 10 ^"8 M or less, such as about 10 ^"9 M or less, for example about 10 ^"10 M or less, or even about 10 ^"11 M or even less, when measured as apparent affinities based on IC ₅ o values.

Preferably said antibody is a naturally occurring antibody or a functional homologue thereof. A naturally occurring antibody is a heterotetrameric glycoprotein capable of recognising and binding an antigen comprising two identical heavy (H) chains and two identical light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises or preferably consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as C _H ). Each light chain comprises, or preferably consists of, a light chain variable region (abbreviated herein as V|_) and a light chain constant region (abbreviated herein as C _L ). The V _H and V _L regions can be further subdivided into regions of hypervariability, termed

complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V _H and V _L comprises and preferably consists of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The naturally occurring antibody may also be a heavy-chain antibody (HCAbs) as produced by camelids (camels, dromedaries and llamas). HCAbs are homodimers of heavy chains only, devoid of light chains and the first constant domain (Hamers- Casterman et al., 1993). Another possibility is New or Nurse Shark Antigen Receptor (NAR) protein, which exists as a dimer of two heavy chains with no associated light chains. Each chain is composed of one variable (V) and five constant domains. The NAR proteins constitute a single immunoglobulin variable-like domain (Greenberg, A. S., Avila, D., Hughes, M., Hughes, A., McKinney, E. C. & Flajnik, M. F. (1995) Nature (London) 374, 168-173.) which is much lighter than an antibody molecule.

Naturally occurring antibodies according to the invention may consist of one heterotetramer or they may comprise several identical heterotetramers. Thus, the naturally occurring antibody according to the invention may for example be selected from the group consisting of IgG, IgM, IgA, IgD and IgE. The subunit structures and three-dimensional configurations of these different classes of immunoglobulins are well known.

Naturally occurring antibodies according to the invention may be antibodies of a particular species, for example the antibody may be a murine, a rat, a rabbit, a goat, a sheep, a chicken, a donkey, a camelid or a human antibody. The antibody according to the invention may however also be a hybrid between antibodies from several species, for example the antibody may be a chimeric antibody, such as a humanised antibody. The antibody according to the invention may be a monoclonal antibody, such as a naturally occurring monoclonal antibody or it may be polyclonal antibodies, such as naturally occurring polyclonal antibodies. The antibody may be any protein or polypeptide containing an antigen binding site, such as a single polypeptide, a protein or a glycoprotein. Preferably, the antigen binding site comprises at least one CDR, or more preferably a variable region.

Thus the antigen binding site may comprise a V _H and/or V _L . In an antibody, the V _H and V|_ together may contain the antigen binding site, however, either one of the V _H or the V|_ may comprise an antigen binding site. In particular, the CDRs may identify the specificity of the antibody and accordingly it is preferred that the antigen binding site comprises one or more CDRs, preferably at least 1 , more preferably at least 2, yet more preferably at least 3, even more preferably at least 4, yet more preferably at least 5, even more preferably 6 CDRs. It is preferable that the antigen binding site comprises at least one CDR3, more preferably at least the CDR3 of the heavy chain.

The antibody may for example be an antigen binding fragment of an antibody, preferably an antigen binding fragment of a naturally occurring antibody, a

heterospecific antibody, a single chain antibody or a recombinant antibody. An antibody according to the invention may comprise one or more antigen binding sites. Naturally occurring heterotetrameric antibodies comprises two antigen binding sites.

It is also not always desirable to use non-human antibodies for human therapy, and accordingly, the antibody of the invention may be a human antibody or a humanised antibody. Thus, the antibody according to the invention may be a human or a humanised antibody. A human antibody as used herein is an antibody, which is obtained from a system using human immunoglobulin sequences. Human antibodies may for example be antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom. Human antibodies may also be isolated from a host cell transformed to express the antibody, e.g., from a transfectoma. Human antibodies may also be isolated from a recombinant, combinatorial human antibody library. The antibody according to the invention may be a chimeric antibody, i.e. an antibody comprising regions derived from different species. The chimeric antibody may for example comprise variable regions from one species of animal and constant regions from another species of animal. For example, a chimeric antibody can be an antibody having variable regions which derive from a mouse monoclonal antibody and constant regions which are human. Such antibodies may also be referred to as humanised antibodies.

Thus, the antibody according to the invention may also be a humanised antibody, which is encoded partly by sequences obtained from human germline immunoglobulin sequences and partly from other sequences. Said other sequences are preferably germline immunoglobulins from other species, more preferably from other mammalian species. In particular a humanised antibody may be an antibody in which the antigen binding site is derived from an immunoglobulin from a non-human species, preferably from a non-human mammal, e.g. from a mouse or a rat, whereas some or all of the remaining immunoglobulin-derived parts of the molecule is derived from a human immunoglobulin. The antigen binding site from said non-human species may for example consist of a complete V _L or V _H or both or one or more CDRs grafted onto appropriate human framework regions in V _L or V _H or both. Thus, in a humanized antibody, the CDRs can be from a mouse or rat monoclonal antibody and the other regions of the antibody are of human origin.

Antigen binding fragments of antibodies

Antigen binding fragments of antibodies are fragments of antibodies retaining the ability to specifically bind to an antigen. Preferably, said fragment is an antigen binding fragment of a naturally occurring antibody.

It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen binding fragments of naturally occurring antibodies include for example (i) a Fab fragment, a monovalent fragment consisting of the V _L , V _H , C _L and Cm domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V _H and Cm domains; (iv) a Fv fragment consisting of the V|_ and V _H domains of a single arm of an antibody or (v) a dAb fragment (Ward et a/., (1989) Nature 341 :544-546), which consists of a V _H domain. Fab fragments may be prepared by papain digestion. F(ab') ₂ fragments may be prepared by pepsin treatment.

The antigen binding fragment of an antibody preferably comprise at least one complementarity determining region (CDR) or more preferably a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.

A further example of an antigen binding fragment of an antibody is binding-domain immunoglobulin fusion proteins comprising (i) an antigen binding site fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The antigen binding site can be a heavy chain variable region or a light chain variable region. Such binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/01 18592 and

US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The antigen binding fragment of an antibody may also be diabodies, which are small antibody fragments with two antigen-binding sites. Diabodies preferably comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 ; and Hollinger et al., Proc. Natl. Acad Sci. USA 90: 6444-6448 (1993).

Method of producing antibody

In general, methods of producing antibodies are well-known to those of skill in the art. Antibodies can be produced using antigenic epitope-bearing peptides or polypeptides derived from an SLC4 family or SLC9 family member, in particular NBCnl or NHE1 . Antigenic epitope-bearing peptides and polypeptides of the present invention preferably contain a sequence of at least four, or between 5 and 100, such as between 10 and 100, such as between 15 and 80, for example between 15 and 70, such as between 15 and 60, such as between 15 and 60, such as between 15 and 40, such as between 15 and 30 amino acids contained within NBCnl , such as SEQ ID NO: 1 or 2 or 14, 15 and/or 16. However, peptides or polypeptides comprising a larger portion of NBCnl , for example containing from 100 amino acids and any length up to and including the entire amino acid sequence of NBCnl are also useful as antigenic epitope-bearing peptides or polypeptides for producing antibodies of the present invention.

The antibodies of the present invention are preferably produced using peptides or polypeptides comprising a region of an extracellular loop of NBCnl or a fragment thereof, such as at least four, or between 5 and 100, such as between 10 and 100, such as between 15 and 80, for example between 15 and 70, such as between 15 and 60, such as between 15 and 60, such as between 15 and 40, such as between 15 and 30 amino acids thereof. In one embodiment, the antigenic epitope-bearing peptides or polypeptides comprise or consists of:

a) EL2, human and mouse: SPVITFGGLLGEATEGRISAIESLFGASLT (SEQ ID NO: 5)

b) EL3, human:

KLFDLGETYAFNMHNNLDKLTSYSCVCTEPPNPSNETLAQWKKDNITAHNISW RNLTVSECKKLRGVFLGSACGHHGP (SEQ ID NO: 6)

c) EL3, mouse:

KLFHLGEIYAFNMHNNLDELTSYTCVCAEPSNPSNETLELWKRKNITAYSVSW GNLTVSECKTFHGMFVGSACGPHGP (SEQ ID NO: 7)

d) EL4, human: PSPKLHVPEKFEPTHPERGWIISPLGDNPW (SEQ ID NO: 8) e) EL4, mouse: PSPKLHVPEKFEPTDPSRGWIISPLGDNPW (SEQ ID NO: 9) f) EL5, human and mouse: SISHVNSLKVESECSAPGEQPKFLGIREQR (SEQ ID NO: 10)

g) Human:

MERFRLEKKLPGPDEEAVVDLGKTSSTVNTKFEKEELESHRAVYIGVHVPFSK ESRRRHRHRGHKHHHRRRKDKESDKEDGRESPSYDTPSQRVQFILGTEDDD EEHIPHDLFTEMDELCYRDGEEYEWKETARWLKFEEDVEDGGDRWSKPYVA TLSLHSLFELRSCILNGTVMLDMRASTLDEIADMVLDNMIASGQLDESIRENVR EALLKRHHHQNEKRFTSRIPLVRSFADIGKKHSDPHLLERNGEGLSASRHSLR TGLSASNLSLRGESPLSLLLGHLLPSSRAGTPAGSRCTTPVPTPQNSPPSSPS ISRLTSRSSQESQRQAPELLVSPASDDIPTWIHPPEEDLEAALKGEEQKNEEN VDLTPGILASPQSAPGNLDNSKSGEIKGNGSGGSRENSTVDFSKVDMNFMRK IPTGAEASNVLVGEVDFLERPIIAFVRLAPAVLLTGLTEVPVPTRFLFLLLGPAG KAPQYHEIGRSIATLMTDEIFHDVAYKAKDRNDLLSGIDEFLDQVTVLPPGEWD

PSIRIEPPKSVPSQEKRKIPVFHNGSTPTLGETPKEAAHHAGPELQRTGRLFG

GLILDIKRKAPFFLSDFKDALSLQC (SEQ ID NO: 1 1 )

h) Human: ATVLSISHVNSLKVESECSAPGEQPKFLGIREQRVT (SEQ ID NO: 12) i) Human: DRIKLFGMPAKHQPDLIYLRYVPLWKVHIFTVIQLTC (SEQ ID NO: 13) j) NBCn1_EL3h1.1 : [Hz]-HNNLDKLTSYSCVCTEPPNPSNETLAQWKKDNITA- amide (SEQ ID NO: 14)

k) NBCn1_EL3h2.1 : [Hz]-LAQWKKDNITAHNISWRNLTVSECKKLRGVFLGSA- amide (SEQ ID NO: 15).

I) NBCn1_EL3h3.1 : CTEPPNPSNETLAQWKKDNITAHNISWRNLTVSE-amide

(SEQ ID NO: 16).

However, any fragment of any of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 may be also be used, for example a fragment comprising at least four amino acids, such as between 5 and 75, such as between 10 and 75, such as between 15 and 70, for example between 15 and 60, such as between 15 and 60, such as between 15 and 50, such as between 15 and 40, such as between 15 and 30 amino acids thereof. In a preferred embodiment, the antibodies are produced using peptides comprising an extracellular polypeptide region of NBCnl and preferably, the extracellular polypeptide region comprises or consists of a sequence selected from any one of SEQ ID NO: 14, 15 and/or 16 or a fragment thereof, such as a fragment of 5-25, such as 10-25, such as 10-20 amino acids thereof. In a preferred embodiment, the antigenic epitope-bearing peptides or polypeptides are human, such as SEQ ID NO: 5, 6, 8, 10, 1 1 , 12, 13, 14, 15 and 16.

It is desirable that the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided). Moreover, amino acid sequences containing proline residues may also be desirable for antibody production.

Recombinant antibodies of the invention may for example be produced using a synthetic library or by phage display, preferably using the extracellular domains of NBCnl to generate antibodies against extracellular domains of NBCnl . Since selection of recombinant antibodies from phage display libraries and synthetic libraries do not build on immunogenicity principles, but solely on binding ability, it is capable of providing antibodies that bind to a more diverse array of epitopes. For example, antibodies binding to synthetic peptides corresponding to surface exposed regions of NBCnl can be isolated. Next, antibodies binding to cells with high expression levels of NBCnl may be isolated. The first step may follow any conventional selection protocol (e.g. Mandrup OA, Friis NA, Lykkemark S, Just J and Kristensen P. A novel heavy domain antibody library with functionally optimized complementarity determining regions. PLoS One 8: e76834, 2013), and the second step could utilize any selection procedures (e.g. Sorensen MD, Agerholm IE, Christensen B, Kolvraa S and Kristensen P. Microselection-affinity selecting antibodies against a single rare cell in a

heterogeneous population. J Cell Mol Med 14: 1953-1961 , 2010; or Sorensen MD and Kristensen P. Selection of antibodies against a single rare cell present in a

heterogeneous population using phage display. Nat Protoc 6: 509-522, 201 1 ; or

Sorensen MD, Melchjorsen CJ, Mandrup OA and Kristensen P. Raising antibodies against circulating foetal cells from maternal peripheral blood. Prenat Diagn 33: 284- 291 , 2013.). Each selection step produces individual bacterial colonies from which monoclonal (phage) antibodies can be produced. The monoclonal phage antibodies are then typically tested in conventional cell ELISA, where antibody binding is compared between cells expressing high amounts of NBCnl (e.g., breast cancer cells isolated from WT mice) and cells with no or very low NBCnl expression (e.g., breast cancer cells isolated from NBCnl knockout mice). In addition, the antibodies can be tested for binding to purified proteins. Following selection, the top monoclonal phage antibody fragments can be cloned into expression vectors, which comprise the constant regions of the IgG or different expression tags.

Recombinant antibodies may be produced in microbial host organisms, such as bacteria, yeast or cell cultures of cells derived from multicellular organisms. Frequently, Escherichia coli is useful as host organism. Frequently, recombinant antibodies are fragments of naturally occurring antibodies comprising at least one antigen binding site, such as a Fab fragment, a Fv fragment or the recombinant antibody is a scFV.

Recombinant antibodies may be identified using various systems, such as phage display or ribosome display. In a preferred embodiment, the present invention relates to methods of producing antibodies targeting extracellular regions of NBCnl using phage display. The starting point of phage display is usually a library of antibodies, such as single chain antibodies or fragments of naturally occurring antibodies expressed by a phage. Various different kinds of phages are suitable for use in phage display, e.g. M13, fd filamentous phage, T4, T7 or λ phage. Phagemids may also be used, but that usually requires use of a helper phage. Typically, the library comprises in the range of 10 ⁷ to 10 ¹⁵ , such as 10 ⁹ to 10 ¹¹ different phages. The antibodies may be either of native or immune origin. The antibodies of the library may be fused to a phage coat protein (e.g. g3p or g8p) in order to ensure display on the surface. Thus, the antibody

(fragment) may be encoded by a nucleic acid sequence, which is cloned upstream or downstream of a nucleic acid encoding a phage coat protein, which is operably linked to a suitable promoter.

The genomic information coding for antibody e.g. for the antibody variable domains may be obtained from B cells of non-immunised or immunised donors using recombinant DNA technology to amplify the VH and VL gene segments and cloning into an appropriate phage. Synthetic libraries may be prepared by rearranging VH and VL gene segments in vitro and/or by introducing artificial sequences into VH and VL gene segments. For example synthetic libraries may be prepared using a VH and VL gene framework, but introducing into this artificial complementarity determining regions (CDRs), which may be encoded by random oligonucleotides.

The library may also be different libraries, which are then combined in the host cell. Thus, one library may comprise heavy chain sequences, such as the heavy chain Fv fragment or Fab fragment or VH and the other light chain sequences, such as the light chain Fv fragment or Fab fragment VL.

Typically, several rounds of selection, e.g. 2, 3, 4, 5 or 5, such as 2 to 5 or 2 to 4 or 2 to 3 rounds of selection are performed. This may be done by immobilising the antigen, contacting the antigen with the phage and isolation of bound phages. The antigen may be immobilised on any suitable solid surface, such as a plastic surface, beads (such as magnetic beads)

In one embodiment, an antibody useful in the therapeutic methods and uses provided herein can be produced and selected in a method comprising the steps of a) Providing an antibody library, preferably a bacteriophage library, which may comprise in the range of 10 ⁷ to 10 ¹⁵ , such as 10 ⁹ to 10 ¹¹ , different phages b) Providing one or more antigens of a gene product of the SLC4A or SLC9A

families, such as NHE1 or NBCnl antigens, preferably an antigen of an

extracellular region of NBCnl or NHE1 ,

c) Contacting said antibody library with said one or more of said antigens, and d) Selecting antibodies, which bind said one or more of said antigens.

The method may comprise additional rounds of selection, such as 2, 3, 4, 5, 5 rounds of selection or more, where a library consisting of the selected antibodies in step d) are contacted with the antigen and again selecting antibodies, which bind the respective antigen. Thus, in one embodiment, the method comprises repeating steps a) -d) once or twice or more, preferably 1 , 2, 3 times, where the antibody library in step a) of additional rounds corresponds to the antibodies selected in step d) of the previous round.

The antibodies of the library are in one embodiment selected for their ability to bind peptides or polypeptide fragments of a gene product of the SLC4A or SLC9A families, such as NHE1 or NBCnl , in particular extracellular fragments (e.g. a polypeptide region comprising a sequence selected from any one of SEQ ID NO: 5-10 or SEQ ID NO: 14-16 or a fragment thereof), or the full-length protein, preferably natively folded. In a preferred embodiment, the antigens are immobilized on a solid surface, for example on solid beads. The provided antigen may be provided as epithelial organoids, for example, epithelial organoids of breast cancer tissue. Thus, in one embodiment, the antigens are provided as epithelial organoids and suitable antibodies are selected by their binding ability to antigens presented on these epithelial organoids. For example, specific antibodies can be identified based on the different binding abilities to epithelial organoids of breast cancers from e.g. NBCnl KO and WT mice.

Polyclonal antibodies to recombinant protein or isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.), pages 1 to 5 (Humana Press 1992), and Williams et al., "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995). The immunogenicity of a polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full- length molecule or a portion thereof. If the polypeptide portion is "hapten-like," such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, or sheep, an antibody specific for a polypeptide according to the present invention may also be derived from a subhuman primate antibody. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., international patent publication No. WO 91/1 1465, and in Losman et al., Int. J. Cancer 46:310 (1990).

Analysis of antibody

The antibodies or antigen binding fragments provided herein specifically recognize and bind a gene product of the SLC4 or SLC9 families, such as NHE1 or NBCnl antigens, in particular an extracellular polypeptide region. Such antibodies and antigen binding fragment thereof can be produced by known methods and/or methods described elsewhere herein. Suitable antibodies and antigen binding fragment thereof can be tested in a number of different assays. In particular, the binding abilities of an isolated antibody or antigen binding fragment thereof can be determined in an ELISA assay. Thus, in one embodiment, the antibody or antigen binding fragment thereof provided herein is capable of displacing one or more reference antibodies in a competitive ELISA assay.

Moreover, suitable antibodies and antigen binding fragment thereof can be analysed by immunoblotting, typically where isolated organoid membranes are incubated with the candidate antibody as primary antibody, and following washing detected by incubating with a detectable secondary antibody, which is typically conjugated to horseradish peroxidase for detection.

Suitable antibodies and antigen binding fragment thereof can also be analysed by immunohistochemistry. For example paraffin-embedded sections of breast tissue can be used and incubated with the candidate antibodies for NBCnl , and antigen binding can subsequently be detected by incubation with a detectable secondary antibody, which is typically conjugated to horseradish peroxidase. The function of suitable antibodies or antigen binding fragment thereof may also be determined on the basis of their ability to inhibit Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange. Thus, in one embodiment, the antibody or antigen binding fragment thereof provided herein is capable of inhibiting cellular Na7HC0 ₃ ^" co-transport and/or Na7H ⁺ exchange, e.g. NBCnl -mediated Na ⁺ -dependent HC0 ₃ ^" transport or NHE1 mediated Na7H ⁺ exchange. One approach could be to test the ability to inhibit Na7HC0 ₃ ^" co- transport and/or Na7H ⁺ exchange in cells, for example in cancer cells, such as epithelial breast cancer organoids. NBCnl is involved in maintaining cellular pH and in particular in avoiding low pH in cancer cells. Therefore, the function of antibodies and antigen binding fragments thereof in recognizing and binding NBCnl and inhibiting cellular Na ⁺ -dependent HC0 ₃ ^" transport can be determined by measuring intracellular pH in human cells and for example mice cells from NBCnl KO vs. WT mice.

Intracellular pH can also be determined in human cancer cell lines, such as MCF7. In particular, the intracellular pH may be determined in organoids from human and/or mice NBCnl KO vs. WT, preferably in epithelial breast cancer organoids. Antibodies or antigen binding fragments thereof, for which the intracellular pH is decreased when supplied to cells or organoids, are functional as NBCnl inhibitors, and are

consequently suitable for use in medicine, in particular in anticancer therapy as described elsewhere herein. Intracellular pH (pH,) can be determined as described in the example. In particular, pH, can be determined as recovery of pH, from acidosis in isolated organoids loaded with BCECF-AM. Epifluorescence is typically measured with a camera-based fluorescence imaging system during alternating excitation at approximately 485 and 440 nm. The 485/440 BCECF fluorescence ratio can be converted to pH, using the high-[K ⁺ ] nigericin calibration technique. Intracellular acidification can be induced with the NH ₄ ⁺ prepulse technique, and acid-base transport activities calculated as the product of the pH, recovery rate and the buffering capacity of that same organoid. Intracellular buffering capacity can be calculated based on the change in pH, upon washout of NH ₄ CI with the assumption that NH ₃ is in equilibrium across cell membranes.

Administration

The therapeutic methods and uses provided herein may involve administration or provision of a therapeutic agent, in particular modulators of Na7HC0 ₃ ^" co-transport and/or Na ⁺ /H ⁺ exchange to a patient. Such modulators are preferably inhibitors and may be chosen from antibodies, peptides and small molecules, such as cariporide for inhibition of Na7H ⁺ exchange.

In general, suitable methods of administering peptides, antibodies and small molecules, including cariporide, are well-known in the art. Thus, any suitable route of administration may be employed for providing a human being with an effective dose of a peptide, small molecule or an antibody or antigen binding fragment thereof as provided herein. For example, oral, rectal, vaginal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Other examples of administration include sublingual, intravenous, intramuscular, intrathecal, subcutaneous, cutaneous and transdermal administration. In one embodiment the administration comprises inhalation, injection or implantation. The administration of the compound according to the present invention can result in a local (topical) effect or a body-wide (systemic) effect. In a preferred embodiment, the antibody or antigen binding fragment thereof is administered by intravenous injection/infusion.

Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. Preferably compounds of the invention are administered orally or intravenously. The effective dose employed of a peptide, small molecule or an antibody or antigen binding fragment thereof may vary depending on the particular compound, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosage may be ascertained readily by a person skilled in the art.

In one embodiment a peptide, small molecule or an antibody or antigen binding fragment thereof of the present invention is administered at a dosage of from about 0.1 milligram to about 100 milligram per kilogram of body weight. For most large mammals, the total dosage is from about 1 .0 milligrams to about 1000 milligrams, preferably from about 1 milligram to about 50 milligrams. In the case of a 70 kg adult human, the total dose will generally be from about 1 milligram to about 350 milligrams. For a particularly potent compound, the dosage for an adult human may be as low as 0.1 mg. The dosage regimen may be adjusted within this range or even outside of this range to provide the optimal therapeutic response.

The weekly dosage of a peptide, small molecule or an antibody or antigen binding fragment thereof is preferably 4-8 mg/kg bodyweight. However, weekly dosage may vary over the course of treatment. Thus, an effective dose of a peptide, small molecule or an antibody or antigen binding fragment thereof may be administered as an initial loading dose of 4-8 mg/kg bodyweight and subsequent doses of 1 -5 mg/kg bodyweight, where e.g. the initial loading dose is administered in week 1 , and the subsequent doses are administered weekly in the following weeks.

A complete course of treatment should preferably result in a total dose of 20-300 mg/kg bodyweight; more preferred 50-200 mg/kg bodyweight, such as about 1 10 mg/kg bodyweight regardless of the dosing regimen employed.

The effective dose is preferably infused intravenously, for example by infusion in 5-180 minutes, preferably between 10 and 120 minutes, such as between 30 and 90 minutes.

Example

Example 1

In this example, it is evaluated if malignant potential of breast cancer tissue and its receptor expression profile are reflected in the relative importance of different mechanisms of net acid extrusion. In particular, we propose that knowledge regarding the mechanisms of net acid extrusion provides 1 ) prognostic information that can be used to select patients at particular risk of developing metastatic breast cancer disease and 2) a diagnostic tool to identify patients that are most likely to respond to specific types of anti-cancer therapy based on inhibitors of acid-base transporters. Methods

Biopsies of human breast cancer and normal breast tissue were sampled from women undergoing breast-conserving breast cancer surgery at Regionshospitalet Randers, Denmark. The included patients had breast tumors larger than 10 mm, were 35 to 85 years of age, and presented with surgically operable breast cancer diagnosed by triple- test (i.e., clinical examination, mammography, and ultrasonography of the breast combined with fine needle aspiration cytology and/or core needle biopsy). The clinical and pathological characteristics of the included patients were evaluated as part of the standard treatment and pathological evaluation. All patients gave written informed consent, and the procedures for obtaining biopsies and handling sensitive personal information were approved by the Mid-Jutland regional committee on Health Research Ethics (1 -10-72-75-12) and the Danish Data Protection Agency (1 -16-02-191 -16), respectively. Preparation of organoids

Epithelial organoids were isolated from the primary breast cancer and normal breast tissue using the following procedure [6]: tissue samples were cut into 1 mm large pieces in phosphate-buffered saline containing (in mM): 154.2 Na ⁺ , 4.1 K ⁺ , 140.6 CI ^" , 8.1 HP0 ₄ ^2" , and 1.5 H ₂ P0 ₄ ^" ; pH 7.4. The tissue was then transferred to Tissue Culture Flat Tubes (Techno Plastic Products AG) containing advanced DMEM/F12 culture medium (Life Technologies, Denmark) supplemented with 10% fetal bovine serum (Biochrom AG, Germany) and a final concentration of 450 lU/mL collagenase type 3 (Worthington Biochemical Corporation, NJ, USA). The culture flasks were then transferred to a shaking incubator (at -60 revolutions per minute) and left overnight in an atmosphere of 5% C0 ₂ at 37°C. Next, portions of the tissue suspensions were transferred to Eppendorf tubes and organoids were allowed to sediment for 20 minutes in the incubator without shaking. To avoid culture-induced changes in cell function or protein expression patterns, intracellular pH recordings were performed on freshly isolated organoids directly without cell culture.

Intracellular pH measurements

Recovery of intracellular pH from acidosis was studied in freshly isolated organoids loaded for 20 minutes with 5 μΜ BCECF-AM and mounted in a custom-built chamber. Epifluorescence was collected using a Nikon Diaphot 200 microscope with excitation light alternating between 495 nm and 440 nm and emission light collected at 510 nm with a Qimaging Retiga-SRV CCD-based fluorescence imaging systems controlled with VisiView (Visitron Systems, Germany) software. The 495/440 BCECF fluorescence ratio was converted to intracellular pH using the high-[K ⁺ ] nigericin calibration technique [7]. Intracellular acidification was induced with the NH ₄ ⁺ prepulse technique [8] and the subsequent rate of intracellular pH recovery was evaluated with and without

CO2/HCO ₃ ^" , first in the absence and then in the presence of bath Na ⁺ (see e.g., Fig. 1 ).

Assuming equilibration of NH ₃ across cell membranes, intracellular intrinsic buffering capacity was calculated based on the change in intracellular pH upon addition and washout of NH ₄ CI in absence of C0 ₂ /HC0 ₃ ^" . Intracellular intrinsic buffering capacity as function of intracellular pH did not differ significantly between organoids isolated from breast cancer tissue and corresponding normal breast tissue (Fig. 6). Contribution from CO2/HCO ₃ ^" to intracellular buffering capacity was calculated using the equation /?CO ₂ /HCO = 2.3 [HCO ₃ ]j. Acid-base transporter activity was calculated as the intracellular pH recovery rate multiplied by the buffering capacity.

The C0 ₂ /HC0 ₃ ^" -containing salt solution used for intracellular pH recordings had the following composition (in mM): 140 Na ⁺ , 4 K ⁺ , 1 .6 Ca ²⁺ , 1 .2 Mg ²⁺ , 122 CI ^" , 24 HC0 ₃ ^" , 1 .2 S0 ₄ ^2" , 1.18 H ₂ P0 ₄ ^~ , 10 HEPES, 5.5 glucose, and 0.03 EDTA. In Na ⁺ -free solutions, Na ⁺ was substituted with an equimolar amount of /V-methyl-D-glucammonium

(NMDG ⁺ ), except for NaHC0 ₃ , which was replaced with choline-HC0 ₃ . In HC0 ₃ ^~ -free solutions, HC0 ₃ ^~ was substituted with an equimolar amount of Cl ^~ . HC0 ₃ ^~ -containing solutions were aerated with a gas mixture of 5% C0 ₂ balance air, HC0 ₃ ^~ -free solutions were gassed with atmospheric air (nominally C0 ₂ -free); pH was adjusted to 7.4 at 37°C. All solutions contained 5 mM probenecid to inhibit cellular extrusion of BCECF by the organic anion transporter.

Statistics

Data are expressed as mean±SEM, unless otherwise specified. Linear relationships were studied using least-squares linear regression analyses. Effects of two variables on the measured variable were evaluated by two-way ANOVA. A probability value (P value) smaller than 0.05 was considered statistically significant; n equals number of patients. Statistical analyses were performed using Graph Pad Prism 5.02 software. Results

When assessing the patient population as a whole, we find that net acid extrusion in breast cancer tissue is mostly Na ⁺ -dependent (Fig. 1 A) with contribution from both C0 ₂ /HC0 ₃ ^~ -dependent and C0 ₂ /HC0 ₃ ^~ -independent transport mechanisms (Fig.

1 A,B). Na ⁺ ,HC0 ₃ ^" -cotransport is observed as an ability of cells to recover intracellular pH faster and/or at more alkaline intracellular pH levels when C0 ₂ /HC0 ₃ ^" is present during the NH ₄ ⁺ -prepulse experiments (Figure 1A,B). The NaVhT-exchange activity can be evaluated based on the Na ⁺ -dependent intracellular pH recovery in the absence of C0 ₂ /HC0 ₃ ^" (Fig. 1 A,B). Our findings support that Na ⁺ ,HC0 ₃ ^" -cotransport (NBCnl ) and Na7H ⁺ -exchange (NHE1 ) are the major acid-base transporters responsible for net acid extrusion in breast cancer tissue. The upregulated Na ⁺ ,HC0 ₃ ^" -cotransport and Na7H ⁺ - exchange is also reflected in the steady-state intracellular pH, which is elevated in organoids from breast cancer compared to normal breast tissue both in the presence and absence of C0 ₂ /HC0 ₃ ^" (Fig. 1 C).

When the patient group is divided into subgroups based on human epidermal growth factor receptor 2 (HER2, a.k.a. ErbB2) expression profile (Fig. 2), presence of lymph node metastases (Fig. 3), and histopathology (Fig. 4), we find that the mechanisms of intracellular pH regulation— i.e., the C0 ₂ /HC0 ₃ ^" -dependency of net acid extrusion and steady-state intracellular pH— varies substantially between the individual groups of patients.

HER2

Overexpression of HER2 receptors and HER2 gene amplification are important prognostic factors in human breast cancer tissue and determine whether targeted therapy against the HER2 receptor (e.g., the monoclonal antibody trastuzumab) is a therapeutic option. We show here that the intracellular pH regulatory function in breast cancer tissue depends on the HER2 status: HER2-positive breast cancer tissue has a much higher Na ⁺ ,HC0 ₃ ^" -cotransport activity than HER2-negative breast cancer tissue (Fig. 2A,C), and this higher C0 ₂ /HC0 ₃ ^" -dependent net acid extrusion is also reflected in a larger C0 ₂ /HC0 ₃ ^" -dependent elevation of steady-state intracellular pH (Fig. 2A,E). The elevated Na ⁺ ,HC0 ₃ ^" -cotransport activity (Fig. 2B,D) and steady-state intracellular pH (Fig. 2B,E) are not observed in normal breast tissue from women with HER2- positive breast cancer when compared to normal breast tissue from women with HER2- negative breast cancer suggesting that it relies on the increased HER2-activity in the malignant breast tissue.

Lymph node metastases

We investigate the intracellular pH regulatory function in primary breast cancer and normal breast tissue from women with metastatic dissemination— including micro- metastases and individual tumor cell metastases— to the regional lymph nodes (Fig. 3). In breast cancer as well as normal breast tissue from women with lymph node metastases, the contribution from Na ⁺ ,HC0 ₃ ^" -cotransport to net acid extrusion is reduced (Fig. 3A-D); and in congruence, the C0 ₂ /HC0 ₃ ^" -dependent elevation in steady-state intracellular pH is decreased compared to women without lymph node metastases (Fig. 3E).

Histopathology

The majority of patient samples collected for the current project are invasive ductal carcinomas or invasive lobular carcinomas. We observe a much greater Na ⁺ ,HC0 ₃ ^~ - cotransport activity in invasive lobular carcinomas compared to invasive ductal carcinomas (Fig. 4A, B, D, and E) and this difference is also reflected in the more prominent C0 ₂ /HC0 ₃ ^" -dependent increase in intracellular steady-state pH in invasive lobular compared to ductal carcinomas (Fig. 4C, F).

Tumor size

Primary tumors differ in size between patients with breast cancer, and since the growth rate of the tumors could depend on the receptor expression profile and correlate with the presence of lymph node metastases, we next test if the mechanisms of intracellular pH control vary as function of tumor size. We find no significant association between the size of the primary tumor and the regulation of steady-state intracellular pH in the breast cancer tissue (Fig. 5). This finding is consistent with previous findings from mice with chemically-induced breast cancer, where NBCnl expression is elevated in breast cancer compared to normal breast tissue but is independent of primary tumor size [2]. We therefore conclude that the variation in Na ⁺ ,HC0 ₃ ^" -cotransport and Na7H ⁺ - exchange dependency in breast cancer tissue from patients with different receptor expression profiles, histopathologies, and with and without lymph node metastases is not explained by differences in size of the primary tumor. Discussion

The present example shows that the relative contribution from Na ⁺ ,HC0 ₃ ^" -cotransport and Na7H ⁺ -exchange to intracellular pH control can be evaluated in breast tissue biopsies from women with breast cancer. Using the same approach, it will also be possible to assess the contribution from other acid-base transporters (e.g., H ⁺ -

ATPases and monocarboxylate transporters) if appropriate transport inhibitors are applied.

Acid-base transporters are potential targets for anti-cancer therapy and information regarding the relative contribution from different acid-base transporters to intracellular pH control is valuable for identifying patients most likely to respond to pharmacological therapy targeting acid-base transport. In order to develop and take full advantage of targeted therapies against functional proteins, it is crucial that the patients with the greatest dependency on these proteins can be identified. Currently, patients are typically selected for targeted therapy (e.g., anti-HER2 or anti-estrogen) based on evaluation of protein expression or gene amplification whereas functional analyses are rarely employed. Because RNA- and protein expression levels are poor correlates for functional activity, a more qualified prediction of treatment responses can be made based on analyses of the functional contribution of the protein.

By comparing cell functions in breast cancer tissue and normal breast tissue, it is possible to evaluate changes occurring during breast carcinogenesis. In particular, we find that the role of Na ⁺ ,HC0 ₃ ^" -cotransport to intracellular pH control is increased in HER2-positive breast cancer tissue compared to normal breast tissue.

The present example shows that breast cancer tissue from women with lymph node metastases differs in intracellular pH regulatory function from breast cancer tissue obtained from women without lymph node metastases: the contribution from

Na ⁺ ,HC0 ₃ ^" -cotransport to net acid extrusion and intracellular steady-state pH regulation is low in the primary breast cancer tissue and normal breast tissue from women with metastatic dissemination to regional lymph nodes. The difference in intracellular pH regulation in normal breast tissue shows that variation in normal tissue function can predispose to metastatic breast cancer disease. A link between acid-base transporter function and cancer metastasis is possible and it is noted that the net acid extrusion from cells can contribute to local extracellular acidification, which can facilitate degradation of extracellular matrix components. Acid-base transporters can also establish intracellular and extracellular pH gradients between the leading and rear end of migrating cells, which can promote cell migration by coordinating cytoskeletal rearrangement and interaction between cells and the extracellular matrix.

In conclusion, we show that biopsies from women with breast cancer can be used to evaluate the contribution from specific acid-base transport mechanisms to intracellular pH control in normal breast tissue and primary breast carcinomas. This information can be used to

a) select patients most likely to respond to therapy targeted at acid-base transporters, b) treat selected patients with appropriate therapies, which involve modulation of the activities of acid-base transporters, and

c) predict prognosis and risk of breast cancer dissemination based on parallel differences in the intracellular pH regulatory function of breast cancer and normal breast tissue from women at increased risk of metastatic breast cancer development. Example 2

In this second example, additional patient material has been included in order to further evaluate if malignant potential of breast cancer tissue and its receptor expression profile are reflected in the relative importance of different mechanisms of net acid extrusion. In particular, we propose that knowledge regarding the mechanisms of net acid extrusion provides 1 ) prognostic information that can be used to identify patients with or at particular risk of developing metastatic breast cancer disease and 2) a diagnostic tool to identify patients that are most likely to respond to specific types of anti-cancer therapy based on inhibitors of acid-base transporters. Methods

The methods employed in this example are identical to those of example 1. However, statistical analyses were performed using GraphPad Prism 7.03 software.

Results

When assessing the patient population as a whole, we find that net acid extrusion in breast cancer tissue is mostly Na ⁺ -dependent (Fig. 7A) with contribution from both C0 ₂ /HC0 ₃ ^~ -dependent and C0 ₂ /HC0 ₃ ^~ -independent transport mechanisms (Fig.

7A,B). Na ⁺ ,HC0 ₃ ^" -cotransport is observed as an ability of cells to recover intracellular pH faster and/or at more alkaline intracellular pH levels when C0 ₂ /HC0 ₃ ^" is present during the NH ₄ ⁺ -prepulse experiments (Fig. 7A,B). The Na7H ⁺ -exchange activity can be evaluated based on the Na ⁺ -dependent intracellular pH recovery in the absence of CO2/HCO ₃ ^" (Fig. 7A,B). Our findings support that Na ⁺ ,HC0 ₃ ^" -cotransport (NBCnl ) and Na7H ⁺ -exchange (NHE1 ) are the major acid-base transporters responsible for net acid extrusion in breast cancer tissue. The upregulated Na ⁺ ,HC0 ₃ ^" -cotransport and Na7H ⁺ - exchange is also reflected in the steady-state intracellular pH, which is elevated in organoids from breast cancer compared to normal breast tissue both in the presence and absence of C0 ₂ /HC0 ₃ ^" (Fig. 7C).

When the patient group is divided into subgroups based on human epidermal growth factor receptor 2 (HER2, a.k.a. ErbB2 or neu) expression (Fig. 8), estrogen receptor expression (Fig. 9), presence of lymph node metastases (Fig. 10), and histopathology (Fig. 1 1 ), we find that the mechanisms of intracellular pH regulation— i.e., the

C0 ₂ /HC0 ₃ ^" -dependency of net acid extrusion and steady-state intracellular pH— varies substantially between the individual groups of patients.

HER2

Overexpression of HER2 receptors and HER2 gene amplification are important prognostic factors in human breast cancer tissue and determine whether targeted therapy against the HER2 receptor (e.g., the monoclonal antibody trastuzumab) is a therapeutic option. We show here that the intracellular pH regulatory function in breast cancer tissue depends on the HER2 status: HER2-positive breast cancer tissue (Fig. 8A,D) has a much higher Na ⁺ ,HC0 ₃ ^" -cotransport activity than HER2-negative breast cancer tissue (Fig. 8B,E), and this higher C0 ₂ /HC0 ₃ ^" -dependent net acid extrusion is also reflected in a larger C0 ₂ /HC0 ₃ ^" -dependent elevation of steady-state intracellular pH (Fig. 8C). The elevated Na ⁺ ,HC0 ₃ ^" -cotransport activity and steady-state intracellular pH are not observed in normal breast tissue from women with HER2-positive breast cancer (Fig. 8A,C,D) when compared to normal breast tissue from women with HER2- negative breast cancer (Fig. 8B,C,E) suggesting that it relies on the increased HER2- activity in the malignant breast tissue.

Estrogen receptors

Estrogen receptors are usually expressed in breast tissue but may become

downregulated during carcinogenesis. Estrogen receptors can be targeted

therapeutically using estrogen receptor modulators (e.g., tamoxifen). We evaluate whether the intracellular pH regulatory function differ between patients with estrogen receptor positive (90-100% ER ⁺ ) and estrogen receptor negative (0-10% ER ⁺ ) primary breast tumors (Fig. 9). In both groups of patients, C0 ₂ /HC0 ₃ ^~ -dependent and - independent acid-base transport mechanisms contribute to net base uptake (Fig 9A,B,D,E); but at low intracellular pH, Na ⁺ ,HC0 ₃ ^" -cotransport is accentuated in estrogen receptor negative compared to estrogen receptor positive breast cancer tissue. Estrogen receptor expression does not significantly affect steady-state intracellular pH (Fig. 9C).

Lymph node metastases

We investigate the intracellular pH regulatory function in primary breast cancer and normal breast tissue from women with metastatic dissemination— including micro- metastases and individual tumor cell metastases— to the regional lymph nodes (Fig.

10). In primary breast cancer tissue from women with lymph node metastases, the contribution from Na ⁺ ,HC0 ₃ ^" -cotransport to net acid extrusion is reduced (Fig.

10A,B,D,E); and in congruence, the C0 ₂ /HC0 ₃ ^" -dependent elevation in steady-state intracellular pH is decreased compared to women without lymph node metastases (Fig.

10C). A similar, however smaller, effect is observed in the normal breast tissue.

Histopathology

The majority of patient samples collected for the current project are invasive ductal carcinomas or invasive lobular carcinomas. We observe a much greater Na ⁺ ,HC0 ₃ ^~ - cotransport activity in invasive lobular carcinomas compared to invasive ductal carcinomas (Fig. 1 1A,B,D,E) and this difference is also reflected in the more prominent C0 ₂ /HC0 ₃ ^" -dependent increase in intracellular steady-state pH in invasive lobular compared to ductal carcinomas (Fig. 1 1 C).

Tumor size and patient age at diagnosis

Primary tumors differ in size between patients with breast cancer, and since the growth rate of the tumors could depend on the receptor expression profile and correlate with the presence of lymph node metastases, we next test if the mechanisms of intracellular pH control vary as function of tumor size. We find no significant association between the size of the primary tumor and the regulation of steady-state intracellular pH in the breast cancer tissue (Fig. 12A). This finding is consistent with previous findings from mice with chemically-induced breast cancer, where NBCnl expression is elevated in breast cancer compared to normal breast tissue but is independent of primary tumor size [2]. We also investigate whether the regulation of steady-state intracellular pH differs as a function of patient age at diagnosis, but observe again no significant dependency (Fig. 12B). We therefore conclude that the variation in Na ⁺ ,HC0 ₃ ^~ - cotransport and Na7H ⁺ -exchange activity in breast cancer tissue from patients with different receptor expression profiles, histopathologies, and with and without lymph node metastases is not explained by differences in size of the primary tumor or patient age at diagnosis.

Discussion

The present example shows that the relative contribution from Na ⁺ ,HC0 ₃ ^" -cotransport and Na7H ⁺ -exchange to intracellular pH control can be evaluated in breast tissue biopsies from women with breast cancer. Using the same approach, it will also be possible to assess the contribution from other acid-base transporters (e.g., H ⁺ - ATPases and monocarboxylate transporters) if appropriate transport inhibitors are applied.

Acid-base transporters are potential targets for anti-cancer therapy and information regarding the relative contribution from different acid-base transporters to intracellular pH control is valuable for identifying patients most likely to respond to pharmacological therapy targeting acid-base transport. In order to develop and take full advantage of targeted therapies against functional proteins, it is crucial that the patients with the greatest dependency on these proteins can be identified. Currently, patients are typically selected for targeted therapy (e.g., anti-HER2 or anti-estrogen) based on evaluation of protein expression or gene amplification whereas functional analyses are rarely employed. Because RNA- and protein expression levels are poor correlates for functional activity, a more qualified prediction of treatment responses can be made based on analyses of the functional contribution of the protein.

By comparing cell functions in breast cancer tissue and normal breast tissue, it is possible to evaluate changes occurring during breast carcinogenesis. In particular, we find that the role of Na ⁺ ,HC0 ₃ ^" -cotransport to intracellular pH control is increased in HER2-positive breast cancer tissue compared to normal breast tissue.

The present example shows that primary breast tumors from women with lymph node metastases differ in intracellular pH regulatory function from primary breast tumors from women without lymph node metastases: the contribution from Na ⁺ ,HC0 ₃ ^~ - cotransport to net acid extrusion and intracellular steady-state pH regulation is low in the primary breast cancer tissue from women with metastatic dissemination to regional lymph nodes. The difference in intracellular pH regulation in primary breast tumor tissue supports that variation in acid-base transport function can facilitate

development— and therefore be used as a marker— of metastatic breast cancer disease. A link between acid-base transporter function and cancer metastasis is possible and it is noted that the net acid extrusion from cells can contribute to local extracellular acidification, which can facilitate degradation of extracellular matrix components. Acid-base transporters can also establish intracellular and extracellular pH gradients between the leading and rear end of migrating cells, which can promote cell migration by coordinating cytoskeletal rearrangement and interaction between cells and the extracellular matrix.

In conclusion, we show that biopsies from women with breast cancer can be used to evaluate the contribution from specific acid-base transport mechanisms to intracellular pH control in normal breast tissue and primary breast carcinomas. This information can be used to

a) select patients most likely to respond to therapy targeted at acid-base transporters, b) treat selected patients with appropriate therapies, which involve modulation of the activities of acid-base transporters, and

c) predict prognosis and risk of breast cancer dissemination based on the different intracellular pH regulatory function of primary breast cancer tissue from women with metastatic breast cancer.

Sequences

SEQ ID NO: 1

Human NBCnl DNA sequence variant a cloned from breast cancer tissue. The underlined 5 ^' -region indicates a region, which differs from SEG ID NO: 2.

atqqaaagatttcgtetqqagaagaagttacctggtcctgatgaagaagctgttgtg gatcttggcaaaactagctcaact gtgaacaccaagtttgaaaaagaagaactagaaagtcatagagctgtatatattggtgtt cacgtcccgtttagtaaagag agtcgtcggcgtcataggcatcgcggacacaaacatcaccaccggagaagaaaagataaa gaatcagataaagaa gatggacgggaatctccttcttatgatacaccatcccagagagttcagtttatccttggt actgaagatgatgatgaagaac atattccccatgatctcttcacggaaatggatgaactgtgttacagagatggagaagaat atgaatggaaagaaactgct agatggctgaaatttgaagaggatgttgaagatggcggtgaccgatggagtaaaccttat gtggcaactctctctttgcac agtctttttgaactaaggagttgcatcctcaatggaacagtcatgctggatatgagagca agcactctagatgaaatagca gatatggtattagacaacatgatagcttctggccaattagacgagtccatacgagagaat gtcagagaagctcttctgaa gagacatcatcatcagaatgagaaaagattcaccagtcggattcctcttgttcgatcttt tgcagatataggcaagaaacat tctgaccctcacttgcttgaaaggaatggtattttggcctctccccagtctgctcctgga aacttggacaatagtaaaagtgg agaaattaaaggtaatggaagtggtggaagcagagaaaatagtactgttgacttcagcaa ggttgatatgaatttcatga gaaaaattcctacgggtgctgaggcatccaacgtcctggtgggcgaagtagactttttgg aaaggccaataattgcatttgt gagactggctcctgctgtcctccttacagggttgactgaggtccctgttccaaccaggtt tttgtttttgttattgggtccagcgg gcaaggcaccacagtaccatgaaattggacgatcaatagccactctcatgacagatgaga ttttccatgatgtagcttata aagcaaaagacagaaatgacctcttatctggaattgatgaatttttagatcaagtaactg tcctacctccaggagagtggg atccttctatacgcatagaaccaccaaaaagtgtcccttctcaggaaaagagaaagattc ctgtgtttcacaatggatctac ccccacactgggtgagactcctaaagaggccgctcatcatgctgggcctgagctacagag gactggacggctttttggtg gtttgatacttgacatcaaaaggaaagcaccttttttcttgagtgacttcaaggatgcat taagcctgcagtgcctggcctcg attcttttcctatactgtgcctgtatgtctcctgtaatcacttttggagggctgcttgga gaagctacagaaggcagaataagtg caatagagtctctttttggagcatcattaactgggattgcctattcattgtttgctgggc aacctctaacaatattggggagcac aggtccagttctagtgtttgaaaaaattttatataaattctgcagagattatcaactttc ttatctgtctttaagaaccagtattggt ctgtggacttcttttttgtgcattgttttggttgcaacagatgcaagcagccttgtgtgt tatattactcgatttacagaagaggctt ttgcagcccttatttgcatcatattcatctacgaggctttggagaagctctttgatttag gagaaacatatgcatttaatatgcac aacaacttagataaactgaccagctactcatgtgtatgtactgaacctccaaaccccagc aatgaaactctagcacaatg gaagaaagataatataacagcacacaatatttcctggagaaatcttactgtttctgaatg taaaaaacttcgtggtgtattctt ggggtcagcttgtggtcatcatggaccttatattccagatgtgctcttttggtgtgtcat cttgtttttcacaacattttttctgtcttca ttcctcaagcaatttaagaccaagcgttactttcctaccaaggtgcgatcgacaatcagt gattttgctgtatttctcacaatag taataatggttacaattgactaccttgtaggagttccatctcctaaacttcatgttcctg aaaaatttgagcctactcatccaga gagagggtggatcataagcccactgggagataatccttggtggaccttattaatagctgc tattcctgctttgctttgtaccatt ctcatctttatggatcaacaaatcacagctgtaattataaacagaaaggaacacaaattg aagaaaggagctggctatc accttgatttgctcatggttggcgttatgttgggagtttgctctgtcatgggacttccat ggtttgtggctgcaacagtgttgtcaat aagtcatgtcaacagcttaaaagttgaatctgaatgttctgctccaggggaacaacccaa gtttttgggaattcgtgaacag cgggttacagggctaatgatttttattctaatgggcctctctgtgttcatgacttcagtc ctaaagtttattccaatgcctgttctgt atggtgttttcctttatatgggagtttcctcattaaaaggaatccagttatttgaccgta taaaattatttggaatgcctgctaagc atcagcctgatttgatatacctccgttatgtgccgctctggaaggtccatattttcacag tcattcagcttacttgtttggtcctttta tgggtgataaaagtttcagctgctgcagtggtttttcccatgatggttcttgcattagtg tttgtgcgcaaactcatggacctgtgt ttcacgaagagagaacttagttggcttgatgatcttatgccagaaagtaagaaaaagaaa gaagatgacaaaaagaa aaaagagaaagaggaagctgaacggatgcttcaagatgatgatgatactgtgcaccttcc atttgaagggggaagtctc ttgcaaattccagtcaaggccctaaaatatagtcctgataaacctgtgagtgtgaaaata agttttgaagatgaaccaaga aagaaatacgtggatgctgaaacttcattatagaattgaaccaagaggcattatacatat agatatatacatatgtaatgtg tgcgtatcatgtcactatatataagaatattgtatgtcatgctgtttatgtgtgactacc gggtttttaaaagtagt SEQ ID NO: 2

Human NBCnl DNA sequence variant b cloned from breast cancer tissue. The underlined 5 ^' -region indicates a region, which differs from SEG ID NO: 1.

atqqaggctqatqqqqccggcgagcagatqagaccgctactcacccggggtcctgat gaagaagctgttgtggatcttg gcaaaactagctcaactgtgaacaccaagtttgaaaaagaagaactagaaagtcatagag ctgtatatattggtgttcac gtcccgtttagtaaagagagtcgtcggcgtcataggcatcgcggacacaaacatcaccac cggagaagaaaagataa agaatcagataaagaagatggacgggaatctccttcttatgatacaccatcccagagagt tcagtttatccttggtactgaa gatgatgatgaagaacatattccccatgatctcttcacggaaatggatgaactgtgttac agagatggagaagaatatga atggaaagaaactgctagatggctgaaatttgaagaggatgttgaagatggcggtgaccg atggagtaaaccttatgtg gcaactctctctttgcacagtctttttgaactaaggagttgcatcctcaatggaacagtc atgctggatatgagagcaagca ctctagatgaaatagcagatatggtattagacaacatgatagcttctggccaattagacg agtccatacgagagaatgtc agagaagctcttctgaagagacatcatcatcagaatgagaaaagattcaccagtcggatt cctcttgttcgatcttttgcag atataggcaagaaacattctgaccctcacttgcttgaaaggaatggtattttggcctctc cccagtctgctcctggaaacttg gacaatagtaaaagtggagaaattaaaggtaatggaagtggtggaagcagagaaaatagt actgttgacttcagcaag gttgatatgaatttcatgagaaaaattcctacgggtgctgaggcatccaacgtcctggtg ggcgaagtagactttttggaaa ggccaataattgcatttgtgagactggctcctgctgtcctccttacagggttgactgagg tccctgttccaaccaggtttttgtttt tgttattgggtccagcgggcaaggcaccacagtaccatgaaattggacgatcaatagcca ctctcatgacagatgagatt ttccatgatgtagcttataaagcaaaagacagaaatgacctcttatctggaattgatgaa tttttagatcaagtaactgtccta cctccaggagagtgggatccttctatacgcatagaaccaccaaaaagtgtcccttctcag gaaaagagaaagattcctgt gtttcacaatggatctacccccacactgggtgagactcctaaagaggccgctcatcatgc tgggcctgagctacagagg actggacggctttttggtggtttgatacttgacatcaaaaggaaagcaccttttttcttg agtgacttcaaggatgcattaagcc tgcagtgcctggcctcgattcttttcctatactgtgcctgtatgtctcctgtaatcactt ttggagggctgcttggagaagctaca gaaggcagaataagtgcaatagagtctctttttggagcatcattaactgggattgcctat tcattgtttgctgggcaacctcta acaatattggggagcacaggtccagttctagtgtttgaaaaaattttatataaattctgc agagattatcaactttcttatctgtc tttaagaaccagtattggtctgtggacttcttttttgtgcattgttttggttgcaacaga tgcaagcagccttgtgtgttatattactc gatttacagaagaggcttttgcagcccttatttgcatcatattcatctacgaggctttgg agaagctctttgatttaggagaaac atatgcatttaatatgcacaacaacttagataaactgaccagctactcatgtgtatgtac tgaacctccaaaccccagcaat gaaactctagcacaatggaagaaagataatataacagcacacaatatttcctggagaaat cttactgtttctgaatgtaaa aaacttcgtggtgtattcttggggtcagcttgtggtcatcatggaccttatattccagat gtgctcttttggtgtgtcatcttgtttttc acaacattttttctgtcttcattcctcaagcaatttaagaccaagcgttactttcctacc aaggtgcgatcgacaatcagtgattt tgctgtatttctcacaatagtaataatggttacaattgactaccttgtaggagttccatc tcctaaacttcatgttcctgaaaaatt tgagcctactcatccagagagagggtggatcataagcccactgggagataatccttggtg gaccttattaatagctgctatt cctgctttgctttgtaccattctcatctttatggatcaacaaatcacagctgtaattata aacagaaaggaacacaaattgaa gaaaggagctggctatcaccttgatttgctcatggttggcgttatgttgggagtttgctc tgtcatgggacttccatggtttgtgg ctgcaacagtgttgtcaataagtcatgtcaacagcttaaaagttgaatctgaatgttctg ctccaggggaacaacccaagtt tttgggaattcgtgaacagcgggttacagggctaatgatttttattctaatgggcctctc tgtgttcatgacttcagtcctaaagtt tattccaatgcctgttctgtatggtgttttcctttatatgggagtttcctcattaaaagg aatccagttatttgaccgtataaaattat ttggaatgcctgctaagcatcagcctgatttgatatacctccgttatgtgccgctctgga aggtccatattttcacagtcattca gcttacttgtttggtccttttatgggtgataaaagtttcagctgctgcagtggtttttcc catgatggttcttgcattagtgtttgtgcg caaactcatggacctgtgtttcacgaagagagaacttagttggcttgatgatcttatgcc agaaagtaagaaaaagaaag aagatgacaaaaagaaaaaagagaaagaggaagctgaacggatgcttcaagatgatgatg atactgtgcaccttcca tttgaagggggaagtctcttgcaaattccagtcaaggccctaaaatatagtgttgatccc tcaattgttaacatatcagatga aatggccaaaactgcacagtggaaggcactttccatgaatactgagaatgccaaagtaac cagatctaacatgagtcct gataaacctgtgagtgtgaaaataagttttgaagatgaaccaagaaagaaatacgtggat gctgaaacttcattatagaa ttgaaccaagaggcattatacatatagatatatacatatgtaatgtgtgcgtatcatgtc actatatataagaatattgtatgtc atgctgtttatgtgtgactaccgggtttttaaaagtagtgtctggagtttgtaatgagca ccgtggagactatgtatttaatgaa atgctctctttgaagtgaggtacatggttctt

SEQ ID NO: 3

Human NBCnl Peptide sequence (variant a) cloned from breast cancer tissue. The underlined N-terminal region indicates where SEG ID NO: 3 differ from SEG ID NO: 4. MERFRLEKKLPGPDEEAVVDLGKTSSTVNTKFEKEELESHRAVYIGVHVPFSKESRRR HRHRGHKHHHRRRKDKESDKEDGRESPSYDTPSQRVQFILGTEDDDEEHIPHDLFTE MDELCYRDGEEYEWKETARWLKFEEDVEDGGDRWSKPYVATLSLHSLFELRSCILN GTVMLDMRASTLDEIADMVLDNMIASGQLDESIRENVREALLKRHHHQNEKRFTSRIP LVRSFADIGKKHSDPHLLERNGILASPQSAPGNLDNSKSGEIKGNGSGGSRENSTVDF SKVDMNFMRKIPTGAEASNVLVGEVDFLERPIIAFVRLAPAVLLTGLTEVPVPTRFLFLL LGPAGKAPQYHEIGRSIATLMTDEIFHDVAYKAKDRNDLLSGIDEFLDQVTVLPPGEW DPSIRIEPPKSVPSQEKRKIPVFHNGSTPTLGETPKEAAHHAGPELQRTGRLFGGLILDI KRKAPFFLSDFKDALSLQCLASILFLYCACMSPVITFGGLLGEATEGRISAIESLFGASL TGIAYSLFAGQPLTILGSTGPVLVFEKILYKFCRDYQLSYLSLRTSIGLWTSFLCIVLVA T DASSLVCYITRFTEEAFAALICIIFIYEALEKLFDLGETYAFNMHNNLDKLTSYSCVCTE P PNPSNETLAQWKKDNITAHNISWRNLTVSECKKLRGVFLGSACGHHGPYIPDVLFWC VILFFTTFFLSSFLKQFKTKRYFPTKVRSTISDFAVFLTIVIMVTIDYLVGVPSPKLHVP EK FEPTHPERGWIISPLGDNPWWTLLIAAIPALLCTILIFMDQQITAVIINRKEHKLKKGAG Y HLDLLMVGVMLGVCSVMGLPWFVAATVLSISHVNSLKVESECSAPGEQPKFLGIREQ RVTGLMIFILMGLSVFMTSVLKFIPMPVLYGVFLYMGVSSLKGIQLFDRIKLFGMPAKH QPDLIYLRYVPLWKVHIFTVIQLTCLVLLWVIKVSAAAWFPMMVLALVFVRKLMDLCFT KRELSWLDDLMPESKKKKEDDKKKKEKEEAERMLQDDDDTVHLPFEGGSLLQIPVKA LKYSPDKPVSVKISFEDEPRKKYVDAETSL ^* SEQ ID NO: 4

Human NBCnl Peptide sequence (variant b) cloned from breast cancer tissue. The underlined N-terminal region indicates where SEG ID NO: 3 differ from SEG ID NO: 4. MEADGAGEQMRPLLTRGPDEEAVVDLGKTSSTVNTKFEKEELESHRAVYIGVHVPFS KESRRRHRHRGHKHHHRRRKDKESDKEDGRESPSYDTPSQRVQFILGTEDDDEEHI PHDLFTEMDELCYRDGEEYEWKETARWLKFEEDVEDGGDRWSKPYVATLSLHSLFE LRSCILNGTVMLDMRASTLDEIADMVLDNMIASGQLDESIRENVREALLKRHHHQNEK RFTSRIPLVRSFADIGKKHSDPHLLERNGILASPQSAPGNLDNSKSGEIKGNGSGGSR ENSTVDFSKVDMNFMRKIPTGAEASNVLVGEVDFLERPIIAFVRLAPAVLLTGLTEVPV PTRFLFLLLGPAGKAPQYHEIGRSIATLMTDEIFHDVAYKAKDRNDLLSGIDEFLDQVTV LPPGEWDPSIRIEPPKSVPSQEKRKIPVFHNGSTPTLGETPKEAAHHAGPELQRTGRL FGGLILDIKRKAPFFLSDFKDALSLQCLASILFLYCACMSPVITFGGLLGEATEGRISAI E SLFGASLTGIAYSLFAGQPLTILGSTGPVLVFEKILYKFCRDYQLSYLSLRTSIGLWTSF L CIVLVATDASSLVCYITRFTEEAFAALICIIFIYEALEKLFDLGETYAFNMHNNLDKLTS YS CVCTEPPNPSNETLAQWKKDNITAHNISWRNLTVSECKKLRGVFLGSACGHHGPYIP DVLFWCVILFFTTFFLSSFLKQFKTKRYFPTKVRSTISDFAVFLTIVIMVTIDYLVGVPS P KLHVPEKFEPTHPERGWIISPLGDNPWWTLLIAAI PALLCTILIFMDQQITAVIINRKEHKL KKGAGYHLDLLMVGVMLGVCSVMGLPWFVAATVLSISHVNSLKVESECSAPGEQPKF LGIREQRVTGLMIFILMGLSVFMTSVLKFIPMPVLYGVFLYMGVSSLKGIQLFDRIKLFG MPAKHQPDLIYLRYVPLWKVHIFTVIQLTCLVLLWVIKVSAAAVVFPMMVLALVFVRKL MDLCFTKRELSWLDDLMPESKKKKEDDKKKKEKEEAERMLQDDDDTVHLPFEGGSL LQIPVKALKYSVDPSIVNISDEMAKTAQWKALSMNTENAKVTRSNMSPDKPVSVKISF EDEPRKKYVDAETSL ^*

SEQ ID NO: 5

EL2, human and mouse: SPVITFGGLLGEATEGRISAIESLFGASLT

SEQ ID NO: 6

EL3, human:

KLFDLGETYAFNMHNNLDKLTSYSCVCTEPPNPSNETLAQWKKDNITAHNISWRNLTV SECKKLRGVFLGSACGHHGP SEQ ID NO: 7

EL3, mouse:

Unformatted residues are fully conserved, bold strongly conserved, italicized weakly conserved, and underlined without consensus between the mouse and human sequence. KLFHLGEIYAFN MH N N LDELTSYTCVCAEPSN PSN ETLELWKR N ITAYSVSWGN LTV

SECKT HGMFVGSACGPHGP

SEQ ID NO: 8

EL4, human: PSPKLHVPEKFEPTHPERGWIISPLGDNPW

SEQ ID NO: 9

EL4, mouse: PSPKLHVPEKFEPTDPSRGWIISPLGDNPW

SEQ ID NO: 10

EL5, human and mouse: SISHVNSLKVESECSAPGEQPKFLGIREQR

SEQ ID NO: 1 1

human

MERFRLEKKLPGPDEEAWDLGKTSSTVNTKFEKEELESHRAVYIGVHVPFSKESRRR HRHRGHKHHHRRRKDKESDKEDGRESPSYDTPSQRVQFILGTEDDDEEHIPHDLFTE MDELCYRDGEEYEWKETARWLKFEEDVEDGGDRWSKPYVATLSLHSLFELRSCILN GTVMLDMRASTLDEIADMVLDNMIASGQLDESIRENVREALLKRHHHQNEKRFTSRIP LVRSFADIGKKHSDPHLLERNGEGLSASRHSLRTGLSASNLSLRGESPLSLLLGHLLP SSRAGTPAGSRCTTPVPTPQNSPPSSPSISRLTSRSSQESQRQAPELLVSPASDDIPT VVIHPPEEDLEAALKGEEQKNEENVDLTPGILASPQSAPGNLDNSKSGEIKGNGSGGS RENSTVDFSKVDMNFMRKIPTGAEASNVLVGEVDFLERPIIAFVRLAPAVLLTGLTEVP VPTRFLFLLLGPAGKAPQYHEIGRSIATLMTDEIFHDVAYKAKDRNDLLSGIDEFLDQVT VLPPGEWDPSIRIEPPKSVPSQEKRKIPVFHNGSTPTLGETPKEAAHHAGPELQRTGR LFGGLILDIKRKAPFFLSDFKDALSLQC SEQ ID NO: 12

human

ATVLSISHVNSLKVESECSAPGEQPKFLGIREQRVT SEQ ID NO: 13

human

DRIKLFGMPAKHQPDLIYLRYVPLWKVHIFTVIQLTC SEQ ID NO: 14

NBCn1_EL3h1.1 : [Hz]-HNNLDKLTSYSCVCTEPPNPSNETLAQWKKDNITA-amide (SEQ ID NO: 14)

SEQ ID NO: 15

NBCn1_EL3h2.1 : [Hz]-LAQWKKDNITAHNISWRNLTVSECKKLRGVFLGSA-amide (SEQ ID NO: 15). SEQ ID NO: 16

NBCn1_EL3h3.1 : CTEPPNPSNETLAQWKKDNITAHNISWRNLTVSE-amide (SEQ ID NO: 16).